isolation and characterization of pistil...
TRANSCRIPT
ISOLATION AND CHARACTERIZATION OF PISTIL PREDOMINANT
GENES FROM TOMATO
By
CHOONG CHIEH WEAN
Thesis Submitted to the School of Graduate Studies, Universiti Putra Malaysia, in
Fulfilment of the Requirements for the Degree of Doctor of Philosophy
August 2004
ii
Abstract of thesis presented to the Senate of Universiti Putra Malaysia in fulfilmentof the requirements for the degree of Doctor of Philosophy
ISOLATION AND CHARACTERIZATION OF PISTIL PREDOMINANT
GENES FROM TOMATO
By
CHOONG CHIEH WEAN
August 2004
Chairman : Ho Chai Ling, Ph.D.
Faculty : Biotechnology and Biomolecular Sciences
The tomato is an economically and nutritionally important crop that has been
extensively used as a research model system to study plant development. In this
study, genes predominantly expressed in the pistil were identified and isolated in
order to develop an understanding of pistil development and function. Since there
have been several studies done in the past examining pistil development, many genes
have been isolated and identified. As it became progressively more difficult to
identify new genes, alternative approaches have to be employed. In this study,
Suppression Subtractive Hybridization (SSH) was used to isolate and identify novel
genes predominantly expressed in the pistil. From a pistil subtraction cDNA library,
550 clones were isolated and analyzed where 32.5 % of the genes were found to be
predominantly expressed in the pistil through reverse northern screening. All the
putative pistil predominant genes were sequenced and 75 independent sequences
were found, 42 which had no homology to any known genes from the database and
thus appear to encode novel proteins. A collection of genes related to metabolism,
iii
photosynthesis and transcription suggest that during anthesis, tomato pistils are more
biologically active than other floral organs. Some of these tomato pistil predominant
genes included extensin-like protein, ribosomal protein L13 and L13E, sterol-C-
methyltransferase and protein kinase C inhibitor-like protein. Two clones, namely
LePiHAT and LePiXTH, were screened for their full-length coding sequences from
a pistil cDNA library. LePiHAT was found to be 90 % similar to histone
acetyltransferase (HAT) from Arabidopsis thaliana while LePiXTH was 88 %
similar to xyloglucan endotransglycosylase/hydrolase (XTH) from A. thaliana.
LePiHAT was predominantly expressed in pistils but was also expressed in young
stamens, sepals, petals, floral meristems and leaves at lower levels. It was postulated
that LePiHAT might be involved in active chromatin remodelling in the pistil.
LePiXTH on the other hand was upregulated in young and mature pistils, young
stamens and in floral meristems but much lower levels were also detected in the
petals, sepals and leaves. Its expression level was highest in floral meristems,
followed by vegetative meristems and mature pistils. Its predominant expression in
floral meristems, coupled with its localized expression in flower buds suggests its
role in floral growth and differentiation. The expression level of LePiXTH in
stamens decreased before anthesis but in mature pistils, it was localized in the
transmitting tissue that led into the ovary that acts as a channel for pollen tube
growth during fertilization. This suggests its role in aiding fertilization through cell
walls reconstruction (xyloglucans molecular grafting). The expression level of
LePiXTH increased in young fruitlets after anthesis but was not detected in ripe
fruit, indicating a possible function in fruit development but not in fruit ripening.
iv
Abstrak tesis yang dikemukakan kepada Senat Universiti Putra Malaysia sebagaimemenuhi keperluan untuk ijazah Doktor Falsafah
PEMENCILAN DNA PENCIRIAN GEN PISTIL PREDOMINAN DARIPADA
TOMATO
Oleh
CHOONG CHIEH WEAN
Ogos 2004
Pengerusi : Ho Chai Ling, Ph.D.
Fakulti : Bioteknologi dan Sains Biomolekular
Tomato ialah tanaman yang penting dari segi ekonomi dan khasiat pemakanan.
Tomato juga digunakan secara meluas sebagai sistem model kajian perkembangan
tumbuhan. Di dalam projek ini, gen predominan di dalam pistil telah dipencilkan dan
dikenalpasti untuk memahami proses perkembangan pistil. Oleh kerana kajian-kajian
seumpama ini pernah dibuat untuk memahami perkembangan pistil, banyak gen
telah pun dipencil dan dikenalpasti. Oleh kerana pengenalpastian gen-gen baru
semakin sukar, kaedah ‘Suppression Subtractive Hybridization’ (SSH) digunakan
untuk memencil dan mengenalpasti gen-gen baru yang predominan di dalam pistil.
Daripada perpustakaan penolakan pistil yang dibina, 550 klon telah dianalisis dan
32.5 peratus daripada gen-gen didapati predominan di dalam pistil melalui
penyaringan ‘reverse northern’. Semua gen yang disangka predominan di dalam
pistil telah dijujuk dan 75 jenis jujukan diperolehi, di mana 42 daripadanya tiada
mempunyai perhubungan dengan gen-gen yang diketahui di dalam pengkalan data.
Di dalam perpustakaan penolakan tersebut, terdapat koleksi gen-gen yang mana
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berfungsi dalam replikasi DNA, metabolisma, fotosintesis and trankripsi,
menunjukkan pistil tomato adalah lebih aktif dari segi biologi daripada organ bunga
yang lain. Beberapa gen-gen dikenalpasti predominan di dalam pistil tomato ialah
extensin-like protein, ribosomal protein L13 and L13E, sterol-C-methyltransferase
and protein kinase C inhibitor-like protein. Dua klon, iaitu LePiHAT dan LePiXTH,
telah disaring untuk jujukan kod daripada perpustakaan cDNA pistil. LePiHAT
adalah 90 % serupa dengan histone acetyltransferase (HAT) daripada Arabidopsis
thaliana manakala LePiXTH pula 86 % serupa dengan xyloglucan
endotransglycosylase /hydrolase (XTH) daripada A. thaliana. LePiHAT adalah
predominan di dalam pistil tetapi juga dizahir di dalam stamen muda, sepal, petal,
meristem bunga dan daun pada aras yang lebih rendah. Adalah disimpulkan bahawa
LePiHAT mungkin terlibat secara aktif dalam senibentuk-semula kromatin di dalam
pistil. LePiXTH pula dinaik-aturkan di dalam pistil muda dan matang, stamen muda
dan meristem bunga dan juga dikesan di dalam petal, sepal dan daun pada aras yang
jauh lebih rendah. Paras penzahirannya adalah paling tinggi di dalam meristem
bunga, diikuti oleh meristem daun dan pistil matang. Sehubungan itu, penzahirannya
yang predominan di dalam meristem bunga dan tumpuan penzahirannya di dalam
pucuk bunga mencadangkan peranannya untuk pertumbuhan dan pembezaan bunga.
Penzahiran LePiXTH di dalam stamen menurun sebelum antesis tetapi
penzahirannya tertumpu pada lorong pertumbuhan tiub debunga semasa
persenyawaan, lalu mencadangkan peranannya untuk menolong persenyawaan
melalui pembinaan-semula dinding sel. Penzahiran LePiXTH meningkat di dalam
buah muda selepas antesis tetapi tidak dapat dikesan di dalam buah masak
menunjukkan fungsinya di dalam perkembangan buah tetapi tidak di dalam
peranuman buah.
vi
ACKNOWLEDGEMENTS
Firstly, I would like to thank Assoc. Prof. Dr. K. Harikrishna, the founder of
the project and entrusting me with the project. I would also like to thank Dr. Ho Chai
Ling (main supervisor), Assoc. Prof. Dr. Rofina Yasmin Othman and Assoc. Prof.
Dr. Suhaimi Napis for being in my supervisory committee and providing necessary
support. I have to thank Dr. Sharifah and Dr. Meilina from Malaysian Palm Oil
Board (MPOB) for their kind support on part of the project, the latter was also
appreciated for introducing me to the hardcore of molecular biology.
Most of the work in this thesis was carried out in the Genetic Lab,
Department of Biotechnology. I would like to thank the lab technician, Mr. Ong
Choon Hoe for his wonderful hospitality throughout the project. Thanks to those that
helped in the tomato project; Pick Kuen, Siti Suhaila, Joey, Alfred, Yen Yen and
Yang Ping. There was also deep appreciation for the sequencing officers from
Institute of Bioscience (IBS), UPM, i.e. Nancy and Musliyana.
Lastly, I would like to dedicate this thesis to my father and mother, for
making this thesis a reality.
vii
I certify that an Examination Committee met on 26 August 2004 to conduct the finalexamination of Choong Chieh Wean on his degree in Doctor of Philosophy thesis
entitled "Isolation and Characterization of Pistil Predominant Genes from Tomato"
in accordance with Universiti Pertanian Malaysia (Higher Degree) Act 1980 and
Universiti Pertanian Malaysia (Higher Degree) Regulations 1981. The Committeerecommends that the candidate be awarded the relevant degree. Members of the
Examination Committee are as follows:
Norihan Mohd. Saleh, Ph.D.Associate ProfessorFaculty of Biotechnology and Biomolecular Sciences
Universiti Putra Malaysia
(Chairman)
Ho Chai Ling, Ph.D.Lecturer
Faculty of Biotechnology and Biomolecular Sciences
Universiti Putra Malaysia
(Member)
Suhaimi Napis, Ph.D.Associate Professor
Faculty of Biotechnology and Biomolecular SciencesUniversiti Putra Malaysia
(Member)
Rofina Yasmin Othman, Ph.D.Associate Professor
Faculty of Science
Universiti of Malaya
(Member)
Roderick John Scott, Ph.D.Professor
University of BathUnited Kingdom
(Independent Examiner)
GULAM RUSUL RAHMAT ALI, Ph.D.
Professor/Deputy Dean
School of Graduate Studies
Universiti Putra Malaysia
Date:
viii
This thesis submitted to the Senate of Universiti Putra Malaysia and has beenaccepted as fulfilment of the requirements for the degree of Doctor of Philosophy.
The members of the Supervisory Committee are as follows:
Ho Chai Ling, Ph.D.
Faculty of Biotechnology and Biomolecular Sciences
Universiti Putra Malaysia(Chairman)
Rofina Yasmin Othman, Ph.D.
Faculty of ScienceUniversity of Malaya
(Member)
K. Harikrishna, Ph.D.Sime Darby Technology Center
(Member)
Suhaimi Napis, Ph.D.Associate Professor
Faculty of Biotechnology and Biomolecular Sciences
Universiti Putra Malaysia
(Member)
AINI IDERIS, Ph.D.
Professor/DeanSchool of Graduate Studies
Universiti Putra Malaysia
Date:
ix
DECLARATION
I hereby declare that the thesis is based on my original work except for equations and
citations, which have been duly acknowledged. I also declare that it has not beenpreviously or concurrently submitted for any other degree at UPM or other
institutions.
CHOONG CHIEH WEAN
Date:
x
TABLE OF CONTENTS
Page
ABSTRACT iiABSTRAK iv
ACKNOWLEDGEMENTS vi
APPROVAL vii
DECLARATION ixLIST OF TABLES xii
LIST OF FIGURES xiii
LIST OF ABBREVIATIONS xviii
CHAPTER
1 INTRODUCTION 1
2 LITERATURE REVIEW 4
2.1 Tomato 4
2.1.1 As a Research Model System 7
2.2 Floral development 92.3 Gene Expression in Pistils 12
2.3.1 Ovaries and Ovules 18
2.4 Gene isolation 22
2.4.1 Differential Expression 232.4.2 Low Abundance Gene 25
2.4.3 Subtraction and Normalization 27
2.5 Notes of Genes of Interest 33
2.5.1 MYST family Histone Acetyltransferase 332.5.2 Xyloglucan Endotransglycosylase/Hydrolase (XTH) 36
3 MATERIALS AND METHODS 43
3.1 Materials 433.2 Isolation of Total RNA and mRNA 43
3.2.1 Total RNA 43
3.2.2 Messenger RNA 45
3.3 Construction of Libraries 473.3.1 Construction of cDNA library 47
3.3.2 Construction of subtraction library 53
3.4 Isolation of Positive Controls 59
3.4.1 RT-PCR 593.4.2 PCR Product Cloning 61
3.5 Protocol for screening 62
3.5.1 Reverse Northern Analysis 62
3.5.2 Full-length Isolation 653.6 Hybridization Analysis 68
3.6.1 Northern Blot Analysis 70
3.6.2 Southern Blot Analysis 72
3.6.3 Semi-quantitative RT-PCR 763.6.4 Fluorescent In Situ Hybridization 80
xi
4 RESULTS 87
4.1 Isolation of Total RNA 87
4.2 Construction of cDNA Library 89
4.3 Construction of Subtraction Library 914.4 Isolation of Positive Controls 93
4.5 Full-length Isolation of Positive Controls 95
4.6 Reverse Northern Analysis 103
4.7 Full-length Isolation 1064.8 Semi-quantitative RT-PCR 114
4.9 Northern Blot Analysis 120
4.9.1 Preliminary analysis 120
4.9.2 Clone 311 1234.9.3 Clone 473 126
4.10 Southern Blot Analysis 129
4.11 Fluorescent In Situ Hybridization 131
4.11.1 Clone 311 1314.11.2 Clone 473 135
5 DISCUSSION 137
5.1 Screening for Pistil Predominant or Specific Clones 1375.1.1 Reliability of a Subtraction Library 145
5.1.2 Validity of Reverse Northern Analysis 146
5.2 Genes Found In Tomato Pistils 149
5.2.1 Tomato Pistils Are Highly Active 1495.2.2 Pistils Predominant Genes 153
5.2.3 Clone 473 is a Putative Histone Acetyltransferase 156
5.3 Clone 311 is a Putative Xyloglucan Endotransglycosylase
/hydrolase 1615.3.1 Expression Pattern and Family Member 161
5.3.2 Putative Function 167
6 CONCLUSION 178
BIBLIOGRAPHY R.1
APPENDIX A A.1
APPENDIX B B.1
APPENDIX C C.1
APPENDIX D D.1
BIODATA OF THE AUTHOR E.1
xii
LIST OF TABLES
Tables Page
1 The fractionation of cDNA according to molecular sizes and theconstructed library size of the primary and amplified cDNA
library.
90
2 The identities of the putative pistil predominant clonescharacterized by northern blot analysis.
121
3 A list of gene entries for the phylogenetic tree in figure 5.2.2 and
their respective accession numbers.
160
4 Similarities of LePiXTH to several XTHs from A. thaliana
according to groups.
162
5 A list of XTH clones entries for the phylogenetic tree in figure
5.3.1 from tomato (Le) and A. thaliana (At) and their respective
accession numbers.
166
6 Data on the initial sequencing of 20 putative pistil predominant
clones.
C.1
7 The identity of the putative pistil predominant clones fromBLAST search.
C.1
xiii
LIST OF FIGURES
Figures Page
1 Model suggested by Coen and Meyerowitz (1991) on the domainaction of the three classes of organ homeotic genes (illustrated as
A, B and C) on the organ formation of the 4 whorl region of a
floral primordium (1, sepal; 2, petal; 3, stamen; 4, carpel). See
text for details.
11
2 Flow diagram of the Suppression Subtraction Hybridization
Methodology.
31
3 Total RNA (a) and mRNA (b) isolated from tomato pistil and
separated on 1 % (w/v) formaldehyde agarose gel.
88
4 Agarose gel (1.2% (w/v)) was used to separate modified cDNAfragments. Size fractionation was made possible by excising the
cDNA (center) based on the molecular size marker (left) for
further purification and manipulations.
90
5 (a) Secondary PCR amplification of the subtraction fragments
based on Subtractive Subtraction Hybridization method separated
on 1.2 % (w/v) agarose gel for size fractionation. (b) Restriction
digestion of 24 subtracted clones using Rsa I to liberate the DNAinsert from the cloning vector demonstrating the presence of
inserts in clones of variable sizes.
92
6 (a) 3’-RACE PCR of Agamous yielded a visible PCR product ofthe expected size of about 900 bp, (b) Cloning of the putative
Agamous was successful where insert was liberated from the
cloning vector by EcoR I and Xba I digestion, (c) RT-PCR of
Cyclophilin yielded a visible PCR product of the expected size ofabout 350 bp, (d) Cloning of the putative Cyclophilin was
successful where inserts were liberated from the cloning vector by
EcoR I and Xba I digestion, (e) RT-PCR of Pal yielded a visible
PCR product of the expected size of about 750 bp.
94
7 Example of plaque lift hybridization signals for primary and
secondary screenings.
96
8 Protein sequence comparison between the isolated putative
CYCLOPHILIN (CyP) with the CYCLOPHILIN sequence
(M55019) from the database confirming the identity of the
putative clone.
96
9 Protein sequence comparison between the isolated putative
AGAMOUS (TAG1) with the AGAMOUS protein sequence
(L26295) from the database confirming the identity of the putativeclone.
97
xiv
10 Protein sequence comparison between the isolated putative PALwith the PAL protein sequence (M90692) from the database
confirming the identity of the putative clone.
98
11 The nucleotide sequence and the putative translated proteinsequence of clone P2.
101
12 Sequences comparison of clone P2 with 2 other putative proteins
from A. thaliana to demonstrate their similarities.
102
13 Reverse northern analysis of 550 clones with cDNA probes from
pistil, stamen, petal and sepal. Petal cDNA probe produced the
least signals, therefore 2 blots were not analyzed with petal cDNAprobe.
104
14 Four clones with identities known from the initial sequencing of
20 putative pistil predominant clones were used to synthesizeprobes to be hybridized to the subtracted clones. These 4 clones
encoded either housekeeping or highly redundant clones (appear
twice in the initial sequencing of 20 clones). In this way,
redundant clones could be reduced for these 4 clones.
105
15 The nucleotide sequence and the putative translated protein
sequence of clone 311.
107
16 Sequences comparison of clone 311 with xyloglucan
endotransglycosylase homologues from other species showing
similarities of clone 311 with other sequences.
108
17 Sequences comparison of clone 311 with XTHs homologues from
tomato showing similarities of clone 311 with other sequences.
Clone 311 is shown to be a new member of XTH genes from
tomato.
110
18 The nucleotide sequence and the putative translated protein
sequence of clone 473.
112
19 Sequence comparison of clone 473 with histone acetyltransferase
homologues from other species showing similarities of up to 80 %
or more to other histone acetyltransferase homologues.
113
20 Semi-quantitative (semi-q) RT-PCR of clone 311 from total RNA
showed high transcript abundance in the floral meristem, pistils
and 5-6 mm stamens. As a control, the semi-q RT-PCR procedure
was also performed using Cyclophilin (CyP) as an internal control.
Northern hybridization with CyP probe was also done on 5 µg of
total RNA to confirm the validity of semi-q RT-PCR results of
CyP expression.
115
xv
21 Semi-quantitative (semi-q) RT-PCR of clone 311 from mRNAalso showed high transcript abundance in the floral meristems and
pistils. As a control, the semi-q RT-PCR procedure was also
performed using Cyclophilin (CyP) as an internal control.
Northern hybridization with CyP probe was also done on 1 µg ofmRNA to confirm the validity of semi-q RT-PCR results of CyP
expression.
116
22 Semi-quantitative (semi-q) RT-PCR of clone 473 from total RNAshowed no significant predominant expression in various tissue
samples. As a control, the semi-q RT-PCR procedure was also
performed using Cyclophilin (CyP) as an internal control.
Northern hybridization with CyP probe was also done on 5 µg of
total RNA to confirm the validity of semi-q RT-PCR results of
CyP expression.
118
23 Semi-quantitative (semi-q) RT-PCR of clone 473 from mRNA
showed predominant expression in pistil and petal only. As a
control, the semi-q RT-PCR procedure was also performed using
Cyclophilin (CyP) as an internal control. Northern hybridization
with CyP probe was also done on 1 µg of mRNA to confirm the
validity of semi-q RT-PCR results of CyP expression.
119
24 Northern blot analysis of several subtracted clones demonstratedthat most of the clones were pistil predominant except for clone
149. However when the signals were normalized with data from
positive controls, clone 149 can be reclassified to be pistil
predominant. Clone P2 was also demonstrated to be pistilpredominant. Pal and Cyclophilin (CyP) were shown to be evenly
and highly expressed in all the floral organs. Agamous (Tag1) was
demonstrated to be expressed in pistils and stamens only.
122
25 Northern blot analysis on 20 µg of total RNA separated on 1 %
(w/v) agarose gel. Clone 311 was reconfirmed to be highly
expressed in floral meristems, pistils and young stamens. As acontrol, the same blot was also hybridized with a Cyclophilin
(CyP) probe.
124
26 Clone 311 is predominantly expressed in floral meristems (lane2),but is also present at lower level in vegetative meristems (lane 1)
comparable to pistils (lane 3).
125
27 Clone 311’s expression was increased in small fruitlets (withattach styles) (lane 3) from pistils collected at 3 days (lane 1) and
5 days (lane 2) interval after anthesis. Clone 311 was not detected
in ripe tomato fruit (lane 4).
125
xvi
28 Clone 311 orthologues were detected in all the flower buds fromother species from the Solanaceae family. However, clone 311
orthologues were only detected in pistils of all tested Solanum
species and N. tabacum.
127
29 Northern blot analysis on 1 µg of mRNA separated on 1 % (w/v)
agarose gel. Clone 473 was found to be predominantly expressed
in pistils after high stringency washes. As a control, the same blot
was also hybridized with Cyclophilin (CyP) probe as acomparison.
128
30 Southern hybridization analysis showed that clone 311 was a
single copy gene in the tomato genome.
130
31 Clone 311 was seen expressed in the transmitting tract of the style
that led into the ovary along the placenta tissue into the ovule of
the mature pistil.
132
32 Clone 311 was expressed evenly in the less than 3 mm flower bud
but the expression was seen higher in the ovules with darker shade
than other tissues.
133
33 Clone 311 is seen expressed in the transmitting tract of the style,
the ovary and ovules, the tapetum layer of the anther, at the base
of the petal, sepal and stamen, and several locations at the base ofthe 3-4 mm flower bud.
134
34 Sections of pistil hybridized with clone 473 probes showed no
significant differences between the experiment and control at 50 Xmagnification.
136
35 Pie chart highlighting the percentages of pistil predominant
sequences and other sequences identified by using reversenorthern screening.
140
36 Charts depicting different types of sequences, i.e. putatively
identified, unknown protein and no significant homology, thatwere present either with redundancies or no redundancies. The left
chart showed the number of different sequences obtained while the
right chart illustrated the percentages. If we assume that sequences
with redundancies represented higher abundance genes andsequences without redundancies represented low abundance genes,
then the unknown and novel sequences were likely to be low
abundance genes.
141
37 Categorization of putative pistil predominant sequences according
to their functions showing high percentages of sequences were
unknown proteins or sequences with no significant similarity to
the database.
143
xvii
38 A phylogenetic tree showing LePiHAT (*) high homologiesamong plant MYST proteins and distanced from human, yeast and
fruit fly MYST proteins.
160
39 A phylogenetic tree displaying LePiXTH (*) distances andgrouping from other XTHs from A. thaliana and tomato, showing
closest relationship with and a possible orthologue of AtXTH9
from A. thaliana.
165
40 Nucleotide sequences comparison between the isolated putative
cyclophilin (CyP) with the cyclophilin nucleotide sequence
(M55019) from the database confirming the identity of the
putative clone.
D.1
41 Nucleotide sequences comparison between the isolated putative
agamous (TAG1) with the agamous nucleotide sequence (L26295)
from the database confirming the identity of the putative clone.
D.2
42 Nucleotide sequences comparison between the isolated putative
PAL with the PAL nucleotide sequence (M90692) from thedatabase confirming the identity of the putative clone.
D.5
xviii
LIST OF ABBREVIATIONS
AMV-RT - Avian Myeloblastosis Virus Reverse Transcriptase
BCIP - 5-bromo-4-chloro-3-indolyl phosphate
bp - basepair
kbp - kilobasepair
BSA - bovine serum albumin
cfu - colony forming unit
CTAB - hexadecyl (or cetyl) trimethyl ammonium bromide
dATP - deoxyadenine triphosphate
dCTP - deoxycytosine triphosphate
dTTP - deoxythymine triphosphate
dGTP - deoxyguanine triphosphate
ATP - adenine triphosphate
CTP - cytosine triphosphate
UTP - uracil triphosphate
GTP - guanine triphosphate
DEPC - diethyl pyrocarbonate
DIG - Digoxigenin
DMSO - dimethyl sulphoxide
DNA - deoxyribonucleic acid
cDNA - complemetary deoxyribonucleic acid
DNase - nuclease
DTT - dithiothreitol
EDTA - ethylene diamine tetracetate
g - gram
mg - milligram
µg - microgram
HCl - hydrochloric acid
LB - Luria-Bertani
LiCl - lithium chloride
M - molar / molarity
mM - millimolar
µM - micromolar
MgSO4 - magnesium sulfate
ml - milliliter
xix
µl - microliter
N - Normality
NaCl - sodium chloride
NaOH - sodium hydroxide
NBT - nitroblue tetrazolium chloride
ng - nanogram
NTE - NaCl-Tris-EDTA
OD - optical density
PBS - phosphate buffer saline
PCR - Polymerase Chain Reaction
pfu - plaque forming unit
pmole - picomole
RNA - ribonucleic acid
mRNA - messenger ribonucleic acid
rRNA - ribosomal ribonucleic acid
RNase - ribonuclease
rpm - revolution per minute
RT - Reverse Transcriptase
SAAP - streptavidin-alkaline phosphatase conjugate
SDS - sodium dodecyl sulfate / sodium lauryl sulfate
SSC - standard saline citrate
TBS - tris buffer saline
TCA - trichloroacetic acid
TE - Tris-EDTA
Tris - tris[hydroxymethyl]aminomethane
Tris-HCl - tris hydrochloride
U - unit
UV - ultraviolet
V - volt
v/v - volume per volume
w/v - weight per volume
X - times
CHAPTER 1
INTRODUCTION
The tomato is a very well known vegetable crop worldwide where it is
extensively used in cooking, for flavoring, as a garnishing and for use with pasta.
Tomato adds flavor to food and also supplies important nutrition for our everyday
needs. Since tomatoes are popular fruit and in some cases are treated as vegetables,
they provide a large boost to several industries. This is evident especially in the food
industries and bakeries worldwide. Examples of the product offered by these
industries are ketchup sauce, pizza sauce, spaghetti sauce and canned tomato puree.
It also helps to promote other products by serving as a flavoring or a garnishing in
food and bakery products. In 1999, the processed tomato production in the United
States of America was more than 9 million metric tons (USA/USDA/Economic
Research Service, 1999).
In recent nutritional and medical studies, tomato was found to be able to
significantly reduce prostate cancer risks in men if taken daily (Gann et al., 1999;
Arnold and Eckstein, 2001; Chen et al., 2001; Giovannucci, 2002; Giovannucci et
al., 2002; Hadley et al., 2002). Tomato contains one of the most potent antioxidants
among the carotenoids known as lycopene. This gives the tomato its attractive
reddish or orangish appearance and is found in higher amounts in tomato than in any
other fruits. Since humans cannot produce lycopene, it is obtained strictly from the
diet and stored in the internal organs such as the liver, lung, skin, colon, prostrate
gland, etc., at higher concentrations than any other known carotenoid. Besides its
2
ability at reducing prostate cancer and other types of cancer, lycopene was
demonstrated to reduce cardiovascular diseases and strengthen the immune system.
Lycopene has been made available in pills as a supplement and this in turn benefits
the pharmaceutical industry. Lycopene is more concentrated in tomato sauce and
represents the free form of lycopene that is easier to be absorbed by the body
compared to the bounded form from fresh tomato fruits.
Tomato has been used extensively as a research model system as it is easy to
grow, has a short life cycle and is easy to work with. Due to its short life cycle,
tomato is a suitable subject for studying plant development. It is easy to germinate
and fast to flower, which makes tomato a suitable study system for studying both
vegetative and floral development. The tomato flowers are of a reasonable size and
are easy to work with. However despite this, relatively little work has been done on
flower development in tomato.
Floral development in plants is an exciting subject to study because of the
unique developmental processes that occur in flowers. Flowering includes a
combination of developmental processes such as vegetative development,
organogenesis and embryogenesis. Organogenesis of floral organs involving several
homeotic genes has been studied extensively for the past decade. We understand
today the mechanism of floral organs formation in general that involves a family of
transcription factors known as MADS box genes. However there is still a need to
study floral determination from vegetative tissue as it is complicated and may vary
from one plant species to another.
3
There has also been little research on molecular biology of zygotic
embryogenesis despite its importance as a tissue that will eventually develop into
fruit and seeds of economical importance. Several types of fruits are much sought
after that are seedless. Some of these seedless fruits are watermelon, papaya, guava,
banana, pomelo, etc. However seedless tomato is not sought after because the seeds
and the pulp surrounding the seeds provide good taste and texture both in food and
tomato puree. Artificial pollination is performed deliberately to increase seed
numbers and for pulp formation, thus increasing the size of the fruit. Therefore it is
crucial to study very early events in fruit development leading to seed development
such as just prior to and after pollination. By unraveling the complex interaction
between proteins in the pistil, we hope to shed light on the process of fertilization
and fruit formation in this important fruit.
The objectives of this study are to isolate and characterize pistil predominant
genes from tomato to understand the function of the tomato pistil. In order to carry
out the objectives, a pistil cDNA subtraction library was constructed and screened
for pistil predominant genes by reverse northern, followed by sequence analysis and
other molecular characterization techniques such as Southern, northern and in situ
hybridization.
4
CHAPTER 2
LITERATURE REVIEW
2.1 Tomato
The tomato is a dicotyledonous plant and its scientific name is Lycopersicon
esculentum. It belongs to a family known as Solanaceae that includes other plants
from other genus such as tobacco, chili, potato, petunia and eggplant. Tomato has a
diploid chromosomal number of 24 (2n = 24). Today, tomato is planted worldwide
for human consumption. Tomato is generally used as a flavoring and as a garnishing
for home cooking and food industries.
Lycopene is a carotenoid that is present in tomatoes, processed tomato
products and other fruits. It is one of the most potent antioxidants among dietary
carotenoids. Antioxidants are protective agents that inactivate reactive oxygen
damage. Antioxidants such as superoxide dismutase, catalase and glutathione
peroxidase are naturally present within human cells. In addition, antioxidants such as
vitamin E, vitamin C, polyphenols and carotenoids are available from food (Agarwal
and Rao, 2000). In tomato, lycopene is the most prominent (abundant) carotenoid
followed by beta-carotene, gamma-carotene and phytoene as well as several minor
carotenoids. The antioxidant activity of lycopene as well as several other carotenoids
and their abundance in tomatoes makes these foods rich sources of antioxidant
activity (Beecher, 1998).
5
There are over 600 carotenoids found in nature where one, lycopene, is
synthesized by plants and microorganisms but not by animals (Paiva and Russell,
1999; Agarwal and Rao, 2000). It is an acyclic isomer of beta-carotene and is highly
unsaturated containing 11 conjugated and 2 unconjugated double bonds (Agarwal
and Rao, 2000). In humans, the bioavailability of lycopene is greater from tomato
paste than from fresh tomato (Gartner et al., 1997). Lycopene appears to be equally
bioavailable from tomato juice and from supplements used in one study (Paetau et
al., 1998).
In general vegetables, fruits and cooked tomatoes, together with olive oil,
appear to be the nutritional traditions that account for the lower risk of several
important chronic diseases, including coronary heart disease and a number of types
of cancer associated with nutritional traditions, such as breast, colon and prostate
cancer (Weisburger, 2002). In addition, prolonged tomato juice consumption
increased plasma lycopene concentrations without significantly affecting cell-
mediated immunity in well-nourished elderly subjects in a study (Watzl et al., 2000).
Stahl et al. (2001) demonstrated that it is feasible to achieve protection
against UV light-induced erythema by ingestion of a commonly consumed dietary
source of lycopene. Other than that, there was a study which indicated that
consumption of commercial tomato juice increases plasma lycopene levels and the
intrinsic resistance of LDL to oxidation almost as effectively as supplementation
with a high dose of vitamin E, which also decreases plasma levels of C-RP, a risk
factor for myocardial infarction, in patients with diabetes. These findings may be