Understanding the genetic basis of storage root formation along with starch and beta

153 Pages·2008·2.79 MB·English

Understanding the genetic basis of storage
root formation along with starch and beta-
carotene biosynthesis and their inter-relation
in Sweetpotato (Ipomoea batatas LAM.).

Inaugural-Dissertation
zur Erlangung des Doktorgrades
an der Universität für Bodenkultur Wien
Department für Angewandte Pflanzenwissenschaften und Pflanzenbiotechnologie
Institut für Angewandte Genetik und Zellbiologie

Vorgelegt von
Dhairyasheel P. Desai

Wien,October 2008

Gutachter 1 Prof. Josef Glößl, IAGZ.
Gutachter 2 Dr. Doz. Eva Wilhelm, ARCS, Seibersdorf, Austria.

Die vorliegend Arbeit wurde im Austrian Research Centres GmbH – ARC, Seibersdorf,
Bereich Biogenetics and Natural Resources, Abteilung Platform for Integrated Clone
Management (PICME) unter der Betreung von Dr. Kornel Burg erstellt.
I
To my Grandfather……………
I I
Acknowledgements

First of all, I would like to thank god for all He has done to show me this day.

I am greatly thankful to Mag. Silvia Fluch and to Dr. Kornel Burg for giving me the
opportunity to work along with them in PICME, Austrian Research Centres, Seibersdorf,
Austria. Without them I wouldn’t have been writing this acknowledgement. Thanks to
Josef Schmidt for all his help.

A special thanks goes to Dr. Kornel Burg for almost every possible invaluable help
during the course of my research. I think he is the “perfect supervisor” and I was
fortunate to be his student. I hope I have not disappointed him about his expectations
from me. Thank you so much for your guidance and continuous support, especially in
developing critical outlook towards the huge gene expression data and interpretation of the
research results. Your help was most important in preparing this thesis and the manuscripts
within.

I would like to acknowledge my gratitude to Dr. Maria Berenyi for her patient introduction to
most of the laboratory techniques I have learned here. I am very grateful for her step-by-step
guidance throughout this research. Her wide knowledge and experience in this field has been
tremendously helpful in guiding the course of this work.

I would like to express my deepest gratitude to my University Supervisor, Prof. Josef Glößl
for his invaluable guidance and advice throughout my studies at the BOKU. His timely
advice in course work was very benevolent and gave me confidence that I would be able to
complete this PhD thesis in light of so many difficulties. He was never too busy to meet me
for the discussions.

I would like to thank a lot to Michael Stierschneider for his help in Microarray chip spotting
and slide scanning, to Dieter, Ildiko and Stephan for their invaluable help in all
bioinformatics related work, to Karin for her help in the laboratory.
II I
I acknowledge the Austrian Research Centres GmbH- ARC in Seibersdorf, Bioresources
division and CIP (Centro Internacional de la Papa), Lima, Peru, for the financial support of
this project. Special thanks to Mrs Agnes Burg and Mag Silvia Fluch for their delicious cakes
on Monday morning lab meetings. Thanks to my fellow PhD students and friends; Ratri,
Edward, Andreas, Renata, Tania, Priscilla, Nadine, Sohail, Joy, Rara, Pepe, Pervez, Afzhal,
Sutti, Jaykumar, Sanjay, Ruturaj, Sudarshan, Yogesh, Saptarshi and All the Kaisrstrasse-106
community friends. You people made my work and stay in Vienna a lot easier than it would
ever have been.

Most of all I would like to thank my parents for their emotional support and for giving me
the opportunity to pursue a career from which I derive great enjoyment and satisfaction.
My heartiest appreciation goes to my beautiful wife for everything. It’s a great fortune to
have such a supportive, understanding and reassuring wife who have made many
sacrifices to make me see this achievement. I would specially thank my Grandfather who
kept faith in me and who inspired all my professional ambitions. I would like to dedicate
this thesis to him.
I will end up with this infinite gratitude, which cannot possibly cover all the names, with
this short Sanskrit prayer, dedicated to all.

ॐॐॐॐ अअअअससससततततोोोो ममममाााा सससस(cid:9)(cid:9)(cid:9)(cid:9)ममममयययय |
ततततममममससससोोोो ममममाााा (cid:11)(cid:11)(cid:11)(cid:11)ययययोोोोििििततततगगगगमम(cid:14)(cid:14)मम(cid:14)(cid:14) यययय |
मममम(cid:16)(cid:16)(cid:16)(cid:16)ृृृृययययोोोोरररर ््््ममममाााा अअअअममममततततृृृृ ं ं ं ं गगगगममममयययय |
ॐॐॐॐ शशशशाााांंििंंिितततत शशशशाााांिंिंिंितततत शशशशाााांिंिंिंितततत || - बहृदार(cid:25)यक उपिनष(cid:31) 1.3.28

Translation,
O’ Lord, Lead Us From Unreal To Real,
From Darkness To Light,
From Death To Immortality,
Let There Be Peace Peace Peace. - Brihadaranyak Upanishad 1.3.28

IV
Abstract

Sweetpotato is one of the most important root crops in the world and constitutes a major
source of dietary carbohydrate in third world countries. Based on comparative analysis of
gene expression in four stages of storage root formation, this study revealed two distinct
phases of storage root development in Sweetpotato. In general, up regulation of genes
were characteristic for the fibrous phase, while an overall down regulation of genes along
with up regulation of some storage related functions characterised the storage phase.
Activity of several signalling pathways could be monitored but Jasmonic acid and
Transforming Growth Factor signalling dominated the fibrous phase while Brassinolide
signalling was predominant in the storage phase.
Biofortified Sweetpotato with higher starch as well as beta-carotene content is of
principle interest in respect to combating Vitamin A deficiency in developing countries.
In the present study Sweetpotato accessions with diverse starch and beta-carotene
phenotypes were compared to discover differences at gene expression level with respect
to starch and beta-carotene biosynthesis. It was observed that beta-amylase and sucrose
phosphate synthase influence the higher starch biosynthesis phenotype in the storage
roots. Extremely high expression of chromoplast differentiation related DnaJ like protein
and Thioredoxin/Ferredoxin post-translation modification system as well as ROS were
found to be related to high beta-carotene content phenotype. Beta-amylase and DnaJ-like
Protein genes could well be acting synergistically towards amyloplast to chromoplast
conversion indicating that controlling mechanisms related to amyloplast/chromoplast
development and differentiation govern the accumulation of starch and beta-carotene in
Sweetpotato storage root.

V
ZUSAMENFASSUNG
Süßkartoffel (Ipomea batatas) ist eine der bedeutendsten knollenbildenden Pflanzen und
wichtiger Kohlenhydratelieferant in den Ländern der Dritten Welt. Der Nähr- und
Inhaltstoffgehalt der Knollen verschiedener Sorten ist sehr unterschiedlich, und führt
dadurch zu unterschiedlicher Eignung als Nahrungsmittel. So findet man Pflanzen mit
orangen Knollen (i.e. hoher Betakarotin Gehalt) und geringem Stärkegehalt, aber auch
weiße Knollen, deren Stärkegehalt weit über dem Durchschnitt liegt. Eine verbesserte
Süßkartoffel mit höherem Stärke- und Betakarotingehalt ist von großem Interesse in
Bezug auf die Bekämpfung des Vitamin A Mangels in der Ernährung in
Entwicklungsländern.
In der vorliegenden Studie wurden verschiedene Süßkartoffel-Sorten mit
unterschiedlichem Stärke- und Betakarotingehalt auf der Genexpressionsebene
verglichen, mit dem Ziel, Gene zu identifizieren, die für Stärke- und Betakarotingehalt
verantwortlich sind.
Basierend auf vergleichenden Genexpressionsanalysen von vier Entwicklungsstadien
während der Wurzelbildung von der Sekundärwurzel hin zur Wurzelknolle konnten zwei
Phasen der Entwicklung identifiziert werden – i) Versorgungswurzel und ii)
Wurzelknolle als Speicherorgan. Während der Entwicklung der Versorgungswurzel trat
mehrheitlich eine Erhöhung der Genexpression verschiedener Gene auf, während die
Speicherphase durch die mehrheitliche Reduktion der Genexpression der selben Genen
charakterisiert war. Nur Gene die eine Rolle in der Speichefunktion spielen, zeigten in
der Speicherphase eine erhöhte Expression.
Eine Untersuchung der Aktivität von verschiedenen Signalpfaden zeigt, dass
Jasmonsäure- und Wachstumsfaktorensignale in der Versorgungswurzel dominieren
während Signale des Brassinol-Signalweges in der Speicherphase zum Zug kommen.
Darüber hinaus wurde beobachtet, dass Beta-Amylase und ‚Sucrose Phosphate Synthase’
die erhöhte Stärkebildung in den Speicherknollen der Phänotypen mit hohem
Stärkegehalt beeinflussen.
Bei Knollen mit hohem Betakarotingehalt konnte eine stark erhöhte Expression der Gene
für die Differenzierung von Chromoplasten, ein DnaJ Protein, sowie Gene für das
‚Thioredoxin/Ferrodoxin post-translations Modifizierungssystem’ und ROS gefunden
V I
werden. Es wird vermutet, dass die Beta-Amylase und das DnaJ Protein bei der
Umwandlung von Amyloplasten zu Chromoplasten synergistisch agieren. Das bedeutet,
dass Kontrollmechanismen während der Amyloplasten/Chromplasten Differenzierung die
Akkumulation von Stärke und Betakarotin in Süßkartoffel-Speicherwurzeln kontrollieren
und steuern.

V II
Table of contents

Acknowledgements.............................................................................................................II
Abstract...............................................................................................................................V
ZUSAMENFASSUNG.....................................................................................................VI
Chapter I..............................................................................................................................1
2.1 General introduction.................................................................................................1
2.1.1 Sweetpotato and beta-carotene..........................................................................2
2.1.2 Sweetpotato and carbohydrate (starch) biosynthesis.........................................3
2.1.3 Storage root formation morphology and molecular biology..............................4
2.1.4 The technology...................................................................................................6
2.1.5 Aims and Objectives..........................................................................................7
CHAPTER 2 Gene expression profiling reveals two distinct phases of storage root
development in Sweetpotato...............................................................................................9
2.1 Abstract...................................................................................................................10
2.1.1 Background......................................................................................................10
2.1.2 Results..............................................................................................................10
2.1.3 Conclusion.......................................................................................................10
2.2 Background.............................................................................................................12
2.3 Results.....................................................................................................................14
2.3.1 General pattern of gene expression during storage root formation..................14
2.3.2 Root developmental stages show two distinct transcriptional profile phases..16
2.3.3 Genes contributing to changing root architecture............................................17
2.3.4 Regulatory processes.......................................................................................17
2.3.5 Signal transduction...........................................................................................18
2.3.6 Growth and development related gene activities.............................................19
2.3.7 Cell differentiation and proliferation related gene activities...........................20
2.3.8 Cell wall metabolism.......................................................................................22
2.3.9 Storage processes.............................................................................................25
2.4 Discussion...............................................................................................................27
2.4.1 Fibrous phase...................................................................................................28
2.4.2 Storage phase...................................................................................................29
2.4.3 Plant defence response during storage root formation.....................................31
2.5 Conclusion..............................................................................................................33
2.6 Material and method...............................................................................................34
2.6.1 cDNA library and Microarray production.......................................................34
2.6.2 Annotations......................................................................................................34
2.6.3 Target preparatio..............................................................................................35
2.6.4 Hybridizations..................................................................................................35
2.6.5 Data analysis....................................................................................................36
VI II
2.6.6 Real-Time PCR analysis..................................................................................36
2.7 Figure legends.........................................................................................................38
2.8 Additional files........................................................................................................39
CHAPTER 3 Elucidation of Starch and Beta-carotene biosynthesis and their inter-relation
in sweetpotato storage roots..............................................................................................53
3.1 Abstract...................................................................................................................54
3.2 Introduction.............................................................................................................55
3.3 Material and method...............................................................................................57
3.3.1 cDNA library and Microarray production.......................................................57
3.3.2 Annotations......................................................................................................57
3.3.3 Target preparation............................................................................................58
3.3.4 Hybridizations..................................................................................................58
3.3.5 Data analysis....................................................................................................59
3.3.6 Real-Time PCR analysis..................................................................................59
3.4 Results.....................................................................................................................61
3.4.1 Global gene expression profile in four sweetpotato accessions.......................61
3.4.2 Starch biosynthesis related gene expression....................................................62
3.4.3 Starch degradation/ related gene expression....................................................64
3.4.4 Beta-carotene biosynthesis related gene expression........................................65
3.4.5 Expression of supporting genes influencing starch and beta-carotene
accumulation.............................................................................................................67
3.5 Discussion...............................................................................................................69
3.5.1 SPS and Beta-amylase controls starch accumulation in sweetpotato..............69
3.5.2 DnaJ-like protein, redox post translation regulatory system and ROS are the
putative regulator of beta-carotene accumulation in sweetpotato storage root.........70
3.6 Figure legends.........................................................................................................74
3.7 Additional files........................................................................................................75
Chapter 4 General conclusion...........................................................................................83
4.1 Sweetpotato storage root development...................................................................83
4.1.1 Fibrous phase...................................................................................................84
4.1.2 Storage phase...................................................................................................84
4.2 Starch and beta-carotene accumulation in storage roots.........................................85
4.2.1 Starch biosynthesis...........................................................................................86
4.2.2 Beta-carotene biosynthesis...............................................................................86
4.2.3 Inter-relation of starch and beta-carotene biosynthesis phenotypes................87
Bibliography.....................................................................................................................91
Annex..............................................................................................................................104

IX
General introduction

Chapter I

2.1
General introduction

Sweetpotato (Ipomoea batatas LAM.) is an important, adaptable, and underexploited food
crop in the world. Annual production of this crop is more than 133 million tons/year and
is cultivated in over 100 developing countries and ranks among the five most important
food crops in over 50 countries (FAOSTAT data, 2005). Sweetpotato currently ranks as
the fifth most important food crop on a fresh-weight basis in developing countries after
rice, wheat, maize, and cassava. The cultivated Sweetpotato is hexaploid (2n=6x=90) and
member of the Convolvulaceae (Morning Glory) family with high level of variations
within the species [Gichuki et al., 2003].
Sweetpotato is principally a small farmer crop and often grown on marginal soils with
limited outputs and production of which tends to be concentrated with lower per capita
income population. It is a natural staple crop of many African countries. The
agronomically important organ of this plant is its tuber, which is actually a modified root
called as storage root which in a form of staple food serves as one of the major sources of
dietary carbohydrates in third world countries. Additionally to carbohydrates Sweetpotato
storage roots also accumulate proteins (Sporamin and Cystatin) and certain genotypes
beta-carotene, which is pro-vitamin to vitamin A. Beta-carotene is an important precursor
for Vitamin-A biosynthesis and its dietary intake is important especially in case of
resource poor in order to fight against Vitamin-A deficiency and its related health issues.
Worldwide, an estimated 250 million under five years’ children are thought to be
deficient in vitamin A [Mason et al., 2001]. In sub-Saharan Africa, some three million
children under the age of five suffer from vitamin A-related blindness. Two-thirds of
affected children die within months of going blind, mainly because of increased
susceptibility to infection associated with insufficient intake of vitamin A [Ruel, 2001].
To make available the biofortified varieties of Sweetpotato to resource poor is an
excellent option to address this issue.

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