Identifikasi Dan Ekspresi Dari Gen Gen Yang Responsif Terhadap Skinning Dan Manfaat Curing Pada Ubijalar

IDENTIFICATION AND EXPRESSION OF SKINNING
INJURY-RESPONSIVE GENES AND CURING PROPERTIES
IN SWEETPOTATO

JOLLANDA EFFENDY

SCHOOL OF GRADUATE STUDIES
BOGOR AGRICULTURAL UNIVERSITY
BOGOR
2015

DECLARATION OF ORIGINALITY AND AUTENTICITY
INCLUDING TRANSFER OF COPYRIGHT*
This is to declare that the dissertation titled “Identification and Expression of
Skinning Injury-Responsive Genes and Curing Properties in Sweetpotato” is the
result of my original research under the direction of the supervisory committee and
that no part of this dissertation has not been submitted for a higher degree to any
other University or Institution. Any other sources of information that have been
mentioned in this dissertation from published or unpublished works of other authors
are fully acknowledged in accordance with the standard reference practices.
Based on this assertion, I hereby transfer the copyright of this dissertation to

Bogor Agricultural University
Bogor, August 2015
Jollanda Effendy
NRP A263100021

RINGKASAN
JOLLANDA EFFENDY. Identifikasi dan Ekspresi dari Gen-gen yang Responsif
Terhadap Skinning dan Manfaat Curing pada Ubijalar. Dibimbing oleh DARDA
EFENDI, NURUL KHUMAIDA, GUSTAAF ADOLF WATTIMENA, dan DON
R. LA BONTE.
Kehilangan kulit dari permukaan umbi bertanggungjawab terhadap kehilangan
hasil pascapanen yang signifikan akibat penyakit pada tempat penyimpanan dan
kehilangan bobot karena respirasi dan transpirasi yang berlebihan. Sayangnya, tidak
ada laporan tentang gen-gen yang terlibat dalam penyembuhan luka pada ubijalar
dan pengetahuan yang lebih mendalam tentang penyembuhan luka akan
memfasilitasi perbaikan strategi pemuliaan. Sistim Annealing control primer
(ACP) digunakan untuk mengidentifikasi gen-gen yang terekspresi setelah
kerusakan akibat skinning (kulit yang terkelupas) dari ubijalar kultivar LA 07-146.
Total didapati 70 gen yang terekspresi berbeda (DEG) yang dapat direproduksi.
Dari 70 DEG ini, 58 terinduksi dan 12 tereduksi. Empat puluh dua diklon dan dari

250 klon yang disolasi, 119 klon dikirim untuk disekuensing. Dari 119 klon ini, 101
klon sama dengan DEG dari tanaman. Kelompok DEG tersebut mewakili 63
unigen: 19 kontig (sekuens dengan tumpang tindih sekurang-kurangnya 50
nukleotida) dan 44 singleton (tidak dapat dibentuk menjadi kontig). Fungsi anotasi
dari DEG menunjukkan gen-gen yang terlibat dalam protein yang berhubungan
dengan stress dan pertahanan, penyandian redoks, metabolism, sintesis protein dan
lokasi akhir protein, regulasi dan signal transduksi.
Perubahan ekspresi gen akibat adanya kerusakan akibat skinning yang
berhubungan dengan 18 DEG dipelajari lebih lanjut. Ke-18 DEG ini menyandikan
gen-gen yang terlibat dalam respons terhadap stress abiotik, biosintesis lignin dan
suberin, regulasi transkripsi dan penyandian. Penelitian tentang ekspresi dari 18
DEG meliputi studi tentang kuantitatif dan semi kuantitatif transkripsi balik rantai
reaksi polimerase (q/sq RTPCR) dibagi menjadi tiga kategori: gen dengan respon
cepat, gen dengan respon lambat dan gen yang respon tidak beraturan. Hasil
penelitian menunjukkan bahwa gen-gen dengan respon cepat umumnya
berhubungan dengan stress secara umum, gen-gen dari lintasan biosintesis lignin
dan suberin ekspresinya meningkat setelah 8 – 12 jam setelah pelukaan (gen-gen
dengan respon lambat). Gen-gen yang lain menunjukkan regulasi ekspresi yang
meningkat atau menurun tergantung dari waktu pengambilan sampel setelah terjadi
pelukaan yang disengaja.

Efek curing dalam meregulasi gen-gen yang terinduksi karena skinning
pada gen-gen yang responnya karena stres, pelukaan dan gen-gen yang terlibat
dalam sintesis lignin dan suberin juga dipelajari. Curing penting untuk
penyembuhan luka dengan merangsang penyembuhan kulit dan mengurangi resiko
dari infeksi dan pembusukan yang terjadi setelah panen. Pola ekspresi dari gen-gen
yang terlibat dalam stress abiotik (IbELIP3), stres karena pelukaan (gen pra-lignin:
IbTAL), lignin biosintesis (IbPAL, IbCCOMT, dan IbCAD), dan suberin (IbExt)
diinvestigasi pada kultivar ubijalar resisten (LA 10-70) dan rentan (LA 07-146)
terhadap kerusakan karena skinning. Pola ekspresi gen-gen yang terinduksi oleh
skinning pada kondisi curing (suhu 28-29.5 oC dengan kelembaban relatif 85-90%)

dan tanpa curing (suhu 24 ± 1 oC dengan kelembaban relatif RH 50%) pada kultivar
ubijalar resisten (LA 10-70) dan rentan (LA 07-146) dari ubijalar juga diteliti.
Tujuannya adalah untuk mempelajari transkripsi untuk mengerti tentang ketahanan
terhadap mekanisme kerusakan karena skinning yang berasosiasi dengan
biosintesis lignin dan pembentukan suberin pada kedua kultivar. Penelitian ini
menunjukkan bahwa gen-gen ini diregulasi secara berbeda pada kultivar resistan
dan rentan dari ubijalar dengan perbedaan pada waktu induksi pada kondisi curing.
Hasil penelitian ini juga menunjukkan bukti dari aliran signal tranduksi yang
terkordinasi dari gen pada lintasan biosintesis pra- lignin dan lignin. Keseluruhan,

penelitian ini mendemonstrasikan perbedaan besar pada toleransi terhadap skinning
antara kedua kultivar tersebut disebabkan karena kemampuan kultivar resisten
untuk mengatur aktivitas transkripsi yang lengkap antara gen-gen pada lintasan
biosintesis pra-lignin dan lignin selama perlakuan skinning, suatu karakteristik
yang hanya ditemukan pada kultivar yang resisten. Penelitian curing ini juga
menunjukkan regulasi dan ekspresi yang berbeda pada gen-gen pada lintasan
biosintesis lignin dan suberin pada kultivar ubijalar yang menunjukkan perbedaan
kemampuan untuk menyembuhkan luka yang terjadi pada level paska panen untuk
membantu memperbaiki kualitas umbi ubijalar.
Kata Kunci: lignifikasi, suberin, pelukaan, ubijalar, curing.

SUMMARY
JOLLANDA EFFENDY. Identification and Expression of Skinning InjuryResponsive Genes and Curing Properties in Sweetpotato. Supervised by DARDA
EFENDI, NURUL KHUMAIDA, GUSTAAF ADOLF WATTIMENA, and DON
R. LA BONTE.
Loss of the skin from the surface of the roots, is responsible for significant
postharvest loss resulting from storage diseases and weight loss. Unfortunately,
there is no report on the genes involved in wound healing of sweetpotato and a
better understanding will facilitate improved breeding strategies. An annealing
control primer (ACP) system was used to identify genes that expressed after

skinning injury of sweetpotato cultivar LA 07-146 storage roots. In total, 70
unambiguous and reproducible differentially expressed genes (DEGs) were
identified. Of these, 58 were up-regulated and 12 down-regulated. Forty two were
cloned and from 250 total clones isolated, 119 independent clones were sent for
sequencing. Of these, 101 clones were related to plants DEGs. These DEGs
represented 63 unigenes: 19 contigs (assembled sequences that were overlapping
by 50 nt) and 44 singletons (that did not have any assemble into a contig).
Functional annotation of the DEGs represented genes involved defense- and stressrelated proteins, redox signaling, metabolism, DNA, RNA related, and gene
expression, intercellular transport, transport facilitation and transport routes,
cellular communication and signal transduction pathway.
The skinning injury changes in gene expression in genes corresponding to
eighteen DEGs were studied further These DEGs encoded genes involved in abiotic
stress responses, lignin and suberin biosynthesis, and transcriptional
regulation/signaling. The expression study of 18 DEGs through quantitative- and
semiquantitative reverse transcription-polymerase chain reaction in response to
skinning injury in sweetpotato roots were divided into three categories: genes with
early response, genes with late response, and genes with transient expression. The
study showed that lignin and suberin pathways were up-regulated after 8 and 12
hours of skinning. Other genes showed up- or down-regulation in their transcript
abundance depending on the time the storage roots were sampled after intentional

skinning.
The effect of curing in regulating skinning-induced changes in genes involved
in general stress, wound stress, and lignin and suberin biosynthesis was also
investigated. Curing is vitally important for wound healing to encourage the skin to
heal and reduce the risk of postharvest infection and rotting. The expression pattern
of genes involved in abiotic stress (IbELIP3), wounding stress (pre-lignin
biosynthesis gene: IbTAL), lignin biosynthesis (IbPAL, IbCCOMT, and IbCAD),
and suberin (IbExt) were investigated in skinning resistant (LA 10-70) and
susceptible (LA 07-146) cultivars of sweetpotato. The expression patterns of
skinning-induced responsive genes in cured (28-29.5 oC with humidity 85-90%)
and in non-cured (24 ± 1 oC and relative humidity RH 50%) conditions in storage
root of resistance (LA 10-70) and susceptible (LA 07-146) cultivars of sweetpotato
were examined. The purpose was to study for their transcript abundance in order to
understand the tolerance to skinning injury mechanisms associated with lignin
biosynthesis and suberin formation in both cultivars. This study revealed that these

genes were regulated in opposite fashion in skinning resistant- and susceptible
cultivars of sweetpotato with difference in timing of their induction under curing
condition. These results also showed evidence for a coordinated gene signaling
cascade in pre- and lignin biosynthesis pathway. Taken together, this study

demonstrated that major differences in skinning tolerance between these two
cultivars were due to the ability of the skinning resistant cultivars to maintain a
complete transcription activity of pre-lignin and lignin biosynthesis pathways genes
during curing treatment following skinning, characteristics were only observed in
skinning resistant cultivars. This curing research also showed differential regulation
and expression of the genes in lignin and suberin biosynthesis pathways in
sweetpotato cultivars that lead to determination the ability to heal skinning wound
occurred during postharvest level to help improve the quality of storage roots of
sweetpotato.
Keywords: lignification, suberin, wounding, sweetpotato, curing,

© Copyright IPB, 2015
Copyright protected under the law
No part or all of this work may be reproduced without citing the source. Copying
may be done only on the basis of education, research, scientific writing, reports or
review; and which should not cause any prejudice to IPB.

IDENTIFICATION AND EXPRESSION OF SKINNING
INJURY-RESPONSIVE GENES AND CURING PROPERTIES
IN SWEETPOTATO

JOLLANDA EFFENDY

Dissertation
Submitted in partial fulfillment of the requirements for the degree
Doctor of Philosophy
in
Plant Breeding and Biotechnology Study Program

SCHOOL OF GRADUATE STUDIES
BOGOR AGRICULTURAL UNIVERSITY
BOGOR
2015

Examiner during Closed Defense:

Prof Dr Ir Sudarsono, MSc
Dr Ir Trikoesoemaningtyas, MSc

Examiners during Promotion Defense:

Prof Dr Ir Sudarsono, MSc
Dr Satya Nugroho

ACKNOWLEDGEMENTS
Glory be to God Almighty for His everlasting blessings, I was able to finish
writing this dissertation.
I would like to thank my senior supervisor, Dr Darda Efendi for his
supervision, patience, advice and encouragement throughout my study and during
the preparation of this dissertation. A similar appreciation is also addressed to Dr Ir
Nurul Khumaida MSi, Prof Dr G.A. Wattimena, MSc, and Prof. Dr. Don R. La
Bonte for being my supervisory committee, and help shaping my PhD research.
Especially to Dr. La Bonte, thank you for discussing my project and reviewing my
dissertation.
I was grateful to Prof Dr Ir Sudarsono, MSc and Prof Dr Ir Bambang S.
Purwoko as Oral Pre-Qualitifaction Examiners. Prof Sudarson MSc and Dr Ir
Trikoesoemaningtyas MSc to serve as closed examiners. Same appreciation was
given to Prof Dr Ir Sudarsono, MSc and Dr Satya Nugroho to serve as doctoral
promotion examiners. Dr. Yudiwanti Wahyu EK, MS as a chair of Plant Breeding
and Biotechnology major thank you for your kindness. Similar appreciation also

went to Dr. Trikoesoemaningtyas.
I would thank Dr. Baisakh for sharing his knowledge and expertise in
molecular biology, in addition to his guidance, advice, discussion, and help in
editing and revising the manuscripts for publication. I was indebted to Dr. La Bonte
for providing the sweetpotato cultivars LA 07-146 and LA 10-70 to be used in this
research. The project would never have been running without permission from
SPESS LSU AgCenter. Other very important contributions were made by the
member of the Molecular Biology Lab at School of Plant, Environmental, and Soil
Sciences at LSU AgCenter. They were Arnold, Nizar, Bode, Renesh, Lina, Julio,
Ramana, and Andres.
To all the lecturers who taught me during my study in IPB, thank you very
much for sharing the knowledge. To all the employee in the Department of AGH
thank you for your help and good laugh.
I wish to express my sincere gratitude to BPPS for funding my PhD study.
My gratitude to Borlaug Fellowship from USDA-FAS and USDA-NIFA for
providing grants to do research in the United States of America. Also to DIKTI
Funding for a Sandwich Program Fellowship to do research in the USA.
For PBT 2010 thank you for the support and friendship through the 5 years.
Ms. Hesti, thanks for sharing your motorbike with me and to Mr. Ismail thanks for
sharing your skill in powerpoint presentation and good discussions.

I also place on record, my sense of gratitude to one and all who directly or
indirectly have lent their hand in this journey.
Finally, I would like to thank my family who always support me through their
prayers, love, encouragement and understanding.

Bogor, August 2015
Jollanda Effendy

TABLE OF CONTENTS
LIST OF TABLES
LIST OF FIGURES
APPENDICES
1 INTRODUCTION
Research Background
Problem Statement
Research Objectives
Hypotheses
Novelty of the Research
Scope and Framework of the Research
2 LITERATURE REVIEW
Skin Formation in Storage Roots and Wound Healing Processes
Skinning Injury and Postharvest Loss
Physical Factors
Physiological Factors
Biological Factors
Strategies Adopted by Plants to Avoid Skinning Injury
Skinning Injury Induced Changes in Gene Expression
Genes Involved in Lignin and Suberin Biosynthesis Pathways
Genes Involved in Protein Fate
Genes Involved in Cell Wall Modification
Genes Involved in Transcription and Protein Synthesis
Genes Involved in Stress Response and Defense
Molecular Biology as Tools to Study Gene Expression in Response to
Sknning Injury in Storage Roots of Sweetpotato
ACP Technology as a Tool to Isolate Differential Expressed Genes
in Storage Roots of Sweetpotato
Real-time Quantitative Polymerase Chain Reaction as a Tool to
Study the Temporal and Developmental Regulation of the
Expression of Skinning Responsive Genes in Sweetpotato
3 FUNCTIONAL CLASSIFICATION OF SKINNING INJURY
RESPONSIVE GENES IN STORAGE ROOTS OF SWEETPOTATO
INTRODUCTION
MATERIALS AND METHODS
Time and Place of Research
Plant Materials and Skinning Treatment
RNA Isolation
cDNA Preparation and ACP-Based Gene-Fishing PCR
Cloning and Sequencing of DEGs
Nucleotide and Deduced Amino Acid Sequencing Analyses
RESULTS AND DISCUSSION
Effect of Skinning treatment on Storage Root RNA Populations
Cloning and sequencing of skinning injury responsive DEGs
Functional Annotation of Selected DEGs

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CONCLUSIONS
4 IDENTIFICATION AND EXPRESSION OF SKINNING INJURYRESPONSIVE GENES IN SWEETPOTATO1
INTRODUCTION
MATERIALS AND METHODS
Time and Place of Research
Plant Materials and Skinning Treatment
RNA Isolation, cDNA Preparation, and ACP-Based Gene-Fishing PCR
Cloning and Sequencing of DEGs
Semiquantitative Reverse Transcription Polymerase Chain Reaction
(sqRT-PCR) Analyzes of Selected DEGs
Quantitative Reverse Transcription Polymerase Chain Reaction (qRTPCR) DEGs
RESULTS AND DISCUSSION
Isolation of DEGs under Skinning Injury
Transcript Abundance Analysis of DEGs
Transcript Abundance Analysis of Stress-Responsive Genes Involved
in Abiotic Stresses
Transcript Abundance Analysis of Genes Involved in Lignin and
Suberin Biosynthesis
Transcript Abundance Analysis of Genes Involved in Transcriptional
Regulation Signaling
CONCLUSIONS
5 CURING ALTERS THE EXPRESSIN OF SKINNING INJURYINDUCED GENES IN TWO CULTIVARS OF SWEETPOTATO
INTRODUCTION
MATERIALS AND METHODS
Time and Place of Research
Plant Materials, Skinning and Curing Treatment
RNA Isolation and cDNA Preparations
Quantitative Reverse Transcription Polymerase Chain Reaction
(qRT-PCR) Analyzes of Selected DEGs
RESULTS AND DISCUSSION
Expression of an Abiotic Stress-Responsive Gene (IbELIP3)
Expression of a Wound-Response Gene (IbTAL)
Expression of Lignification-Associated Genes
IbPAL
IbCCOMT
IbCAD
Expression of IbExt, a Suberin-Related Gene
CONCLUSIONS
6 GENERAL DISCUSSIONS
7 CONCLUSIONS AND RECOMMENDATIONS
Conclusions
Recommendations
REFERENCES
BIOGRAPHY

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LIST OF FIGURES
Figure 1.1

Research flowchart

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Figure 2.1

Potato native and wound periderm

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Figure 2.2

The monolignol biosynthesis pathway and typical lignin
distribution in monocot Switchgrass and dicot

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Representative gel pictures from PCR with ACP2 with
different primers

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Figure 3.2

Alignment of the deduced amino acid of fifteen DEGs

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Figure 3.3

Distribution of DEGs length and percentage of transcripts
with BLASTX hits
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Figure 3.4

Sequence identity distribution

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Figure 3.5

Top-Hit species distribution

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Figure 3.6

Top-Hit DEGs distribution

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Figure 4.1

Representative gels from PCR with annealing control
primers (ACP)

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Semiquantitative reverse transcription PCR analysis of
differentially expressed genes in storage roots of
sweetpotato

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Expression of differentially expressed genes in storage
root of sweetpotato at 2, 4, 8 and 12 h relative to 0 h
after skinning

50

Expression of ELIP3 (Early light-inducible protein)
IbSIn61a) ) in storage roots of LA 07-146 and
LA 10-70 cultivars of sweetpotato, skinned and
cured (C) (at 28-29.5 oC with RH 85-90%) and
non-cured (NC) (24 ± 1 oC with RH 50%) at 0, 2,
4, 8, 12, and 24 h.

58

Expression of TAL (Transaldolase) (IbSIn46) ) in
storage roots of LA 07-146 and LA 10-70 cultivars
of sweetpotato, skinned and cured (C) (at 28-29.5 oC
with RH 85-90%)
and non-cured (NC) (24 ± 1 oC
with RH 50%) at 0, 2, 4, 8, 12, and 24 h.

59

Expression of PAL (Phenylalanine ammonia lyase)
(IbPAL) ) in storage roots of LA 07-146 and LA
10-70 cultivars of sweetpotato, skinned and cured (C)
(at 28-29.5 oC with RH 85-90%) and non-cured (NC)
(24 ± 1 oC with RH 50%) at 0, 2, 4, 8, 12, and 24 h.

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Figure 3.1

Figure 4.2

Figure 4.3

Figure 5.1

Figure 5.2

Figure 5.3

Figure 5.4

Expression of CCOMT (Caffeoyl-Coenzyme A 3-O-

Figure 5.5

Figure 5.6

methyltransferase) (IbCCOMT) ) in storage roots of
LA 07-146 and LA 10-70 cultivars of sweetpotato,
skinned and cured (C) (at 28-29.5 oC with RH 85-90%)
and non-cured (NC) (24 ± 1 oC with RH 50%) at 0, 2,
4, 8, 12, and 24 h.

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Expression of CAD (Cinnamyl alcohol dehydrogenase)
(IbCAD) in storage roots of LA 07-146 and LA 10-70
cultivars of sweetpotato, skinned and cured (C)
(at 28-29.5 oC with RH 85-90%) and non-cured (NC)
(24 ± 1 oC with RH 50%) at 0, 2, 4, 8, 12, and 24 h.

63

Expression of Ext (Extensin) (IbExt) ) in storage roots
Of LA 07-146 and LA 10-70 cultivars of sweetpotato,
skinned and cured (C) (at 28-29.5 oC with RH 85-90%)
and non-cured (NC) (24 ± 1 oC with RH 50%) at 0, 2,
4, 8, 12, and 24 h.

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LIST OF TABLES
Table 3.1

Accumulation of ACP-gene fishing products corresponding
to RNA from storage root of sweetpotato subjected to
skinning injury
22

Table 3.2

Functional annotation of fifteen DEGs in response to
skinning injury

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Differentially expressed genes (DEGs) induced in
response to skinning

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Genes used for expression profiling under curing and
non-curing conditions of sweetpotato storage root
and their corresponding primer sequences

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Tabel 4.1
Table 5.1

1

1 INTRODUCTION
Research Background
Sweetpotato, the world’s seventh important food crop, is the main staple in
82 developing countries. The world sweetpotato production is estimated to be 8.18
Mt (FAO β01γ). Asia and the Pacific Islands account for 87.γ% of world’s
sweetpotato production. Indonesia is the world’s fourth largest sweetpotato
production in the world with total production 2.386 Mt and total harvested area
0.161MHa with the average yield 14.7 ton/ha.
In Indonesia, sweetpotato is the second most important tuber crops after
cassava. Besides high in starch and sugar, sweetpotato is also high in vitamin C,
provitamin A, vitamin B, (thiamine) and iron. Some variety of sweetpotato are rich
in ß-carotene and anthocyanine. Despite its important as a food crops, research in
sweetpotato in Indonesia have mainly focused on breeding for sweetpotato cultivars
with traits such as: adapted to different climate conditions, improve taste or
nutritional value, better cope with diseases or pests in the field, or to use water or
nutrients more efficiently and high yield production. There is need to improved
postharvest handling and processing of storage roots (Saleh and Hartojo 2003). Due
to its thin and delicate skin, sweetpotato roots are often subjected to skinning during
harvest, transport from field to the market or to storage facilities. Skinning, the loss
of skin (periderm) from the surface of the roots is the main culprit of reduced root
weight by water loss associated with increase rate of moisture and weight loss,
shriveling of the root surface, increase susceptibility to pathogen attack and
unattractive appearance. Skinning injury is unavoidable in developed and
developing countries. In developed countries, skinning injury is due to mechanical
injury, while in developing countries is due to hand-harvested. Skinning tolerance
is thus equally important in developed and developing countries.
Many studies have shown altered gene expression in response to skinning
injury. The rapid induction of mRNA transcripts related to defense and wound
healing may protect plants from attack by fungi and pathogens during storage
(Bowles 1990; Chen et al. 2005). These defense genes were activated through
transcriptional, post-transcriptional and post-translational regulation. Furthermore,
lignin deposition are accompanied by changes in genes involved in lignin
biosynthesis pathway. In addition to the role of lignin as a resistance factor, induced
lignification has been proposed as an active resistance mechanism of plants against
fungi; at sites of wounding or pathogen attack. Lignin formation has been observed,
to strengthen the cell wall at the location of damage (Vance et al. 1980; BorgOlivier and Monties 1993; Hawkins and Boudet 1994). Lignin biosynthesis has
been proposed to be controlled by two signal transduction pathways, one involved
in the development of lignified tissues and the other in plant defense response
(Walter 1992).
No studies have been conducted on breeding of plants with skinning injury
tolerance including physiological, cellular, and molecular responses of plants to
skinning injury.

2

Problem Statement
Sweetpotato is the third most economically important root crops after
potatoes and cassava. It has been estimated that losses in sweetpotato due to
physical wounding, such as skinning, cuts and bruises can be as high as 40%.
Skinning is inevitable due to the rigors of bulk harvesting in sweetpotato, and thus
skinning resistance is a prerequisite to developing sweetpotato that can withstand
subsequent postharvest loss caused by storage disease and insect predicament.
Wound responses in plants have been of interest to researches for many years,
especially in those plants that are of economic and nutritional importance. One of
the main reasons for studying wound response is to gain an understanding of the
processes that plants will undergo to minimize infection and fluid loss after injury.
When plant tissue is damaged, a variety of physiological and anatomical changes
occurs in the cells surrounding the wound. Plant organs are generally protected from
desiccation and infection by pathogens by substances such as cutin, suberin, and
lignin. When injury to surfaces of plants occurs, cells exposed to the environment
may become desiccated and (or) infected unless impermeability is rapidly
reestablished. Wound responses may differ among different plant species and
organs. The ability of plant tissue to heal wounds is vital to prevent excessive water
loss and pathogen invasion. This is exploited to improve storability of root crops
after harvest by a process termed curing, in which they are placed in an environment
to promote healing of wounds incurred during harvesting and handling.
Understanding the genetics of wound healing through detail analysis of
specific genes in different regulatory/signaling pathway of sweetpotato will help
breeding for storage roots with tough skin acquiescent to mechanized harvesting in
the developed countries while in developing countries, tough skin may help
extended marketing shelf life and also reduce postharvest losses.

Research Objectives
The major objective of my research was to isolate, identify, and characterize
novel skinning injury responsive genes in sweetpotato storage roots and to examine
their expression when subjected to skinning injury. A further objective was to
elucidate the role of curing on the expression of skinning injury responsive genes
in two cultivars of sweetpotato in order to determine if curing treatment mediates
changes in gene expression of skinning injury responsive genes.
1.
The objective of Experiment I was to isolate, identify and characterize the
function of the genes/transcripts that are responsive to skinning injury due to
intentional skinning in storage roots of sweetpotato.
2.
The objective of Experiment II was to understand the temporal and
developmental regulation of the expression of skinning-responsive genes.
3.
The objective of Experiment III was to differentiate wound healing efficiency
in response to curing due to skinning in skinning-injury resistant and –
susceptible cultivars of sweetpotato.

3

Hypotheses
1.
2.
3.

This research were based on several assumptions:
There were up-regulation and down-regulation of differentially gene
expressions in response to skinning injury in storage roots of sweetpotato.
There were differences in the level and time of gene expressions in response
to skinning injury in storage roots of sweetpotato.
There were differences in the expression of genes induced during curing due
to skinning in skinning injury resistant and -susceptible cultivars of
sweetpotato

Novelty of the Research
Research in sweetpotato is not as advance as research in potato. However,
until now, no molecular data exists for skinning in potato as well. Due to its delicate
and thin skin (periderm), sweetpotato is prone to several forms of postharvest losses
during transportation from the field and in storage. Furthermore, skinning cause an
increase in moisture and weight losses. Curing is pre-requisite to alleviate
postharvest loss due to the loss of skinning from the surface of the roots. Gene
expression studies in sweetpotato still focus on the aspect of understanding storage
root formation and development, transcription profiling in storage root formation
and lignin and starch biosynthesis, initiation of storage root development, sucrose
metabolism, (a)biotic stress responses. No study outside the present work have
addressed directly the effect of curing on the induction of skinning injury responsive
genes. This dissertation presents experimental research that is unique:
1.
Identified, characterized and classified the functions of skinning injury
responsive genes are identified, characterized and classified.
2.
Studied the spatial and temporal expression of skinning injury responsive
genes in a time course manner.
3.
Investigated the effect of curing on the expression of genes involved in
general stress response, wound inducible response, lignin and suberin
biosynthesis pathways in skinning injury resistant and –susceptible cultivars
of sweetpotato.
4.
Elucidated the role of TAL (pre-lignin) gene in lignin biosynthesis pathway.
5.
Assessed the role of CAD as the final key step in lignin biosynthesis pathways
important for lignin formation.
6.
Made a recommendation of using cinnamyl alcohol dehydrogenase (CAD)
gene as a molecular marker specific for lignin biosynthesis.
7.
Differentiated the role of early (short term rapidly induced) and late gene
response with respect to wound healing in response to skinning injury.
8.
Determined that the genes identified in this research are not limited to study
skinning injury in sweetpotato but also apply to all stems, fruits, and roots of
dicotyledons and gymnosperms in relation to skinning/wounding.

4

Scope and Framework of the Research
This research is consisted of three experiments. The first stages is to isolate,
identify, and characterize the functions of the genes/transcripts that are responsive
expression of eighteen selected genes that are induced during skinning injury. The
last stage is to study the expression of six selected genes under cured and non-cured
condition in susceptible- and resistant cultivars of sweetpotato (Figure 1.1).
In Experiment I, skinning injury induced changes in gene expression were
analyzed at mRNA level using ACP-based DD-RTPCR). This experiment began
with isolation of total RNA from sweetpotato cultivar LA 07-146 from three
independent roots subjected to skinning at 0 (control), 2, 4, 8 and 12 hr. The next
step was to performe cDNA preparation and ACP-based gene fishing PCR.
Following agarose gel electrophoresis, the selected bands of interest were excised
from the gel, extracted using Qiaquick Gel extraction kit and then cloned into
pGEM®-T Easy Vector (Promega). After removing the vector backbone and poly
(A), DEGs were sent for sequencing. Functional classification of DEGs was
performed by compared against all sequences in the non redundant database at
NCBI using BlastN and BlastX (Altschul et al. 1997). These distinct identifications
were grouped into their functional categories.
In Experiment II, Quantitative and semi quantitative Reverse TranscriptionPolymerase Chain Reaction q(sq) RT-PCR were used to study the expression of
skinning induced genes. Fifteen selected DEGs from Experiment I and three
additional genes (lignin and suberin related genes) were selected and primers were
designed using Primer3.
In potato, curing has been shown to induce changes in a(biotic)-stress-related
genes. In present study, the role of curing in regulating the selected skinning
responsive expression of gene(s) corresponding to skinning responsive cDNA was
determined:
1.
By subjecting roots to curing and not-curing treatments after skinning
treatments at 0, 2, 4, 8, 12 and 24 h.
2.
Using skinning injury resistant- and susceptible cultivars of sweetpotato.
3.
The flow chart of this present research is presented in Figure 1.1.

5

Experiment I:

Identification of Genes/Transcripts that are Responsive
to Wound Injury due to Skinning in Storage Roots
of Sweetpotato
Isolation of Total RNA from Storage Roots of Sweetpotato

cDNA Preparation and ACP-Based Gene Fishing PCR

Fragments of DEGs

Cloning and Sequencing of DEGs

·
·
·
·
·
·
·
·

Isolate and Elute the DEGs fragments
Gel Extraction of DEGs to elute DNAs
Ligation of DEGs
Transformation of DEGS
Growing the Bacterial Overnight
PCR Bacterial Suspension Cultures
Run the Agarose Gel to Check the Inserts
Sequencing the DEGs

Differentially Expressed Genes (DEGs)

Functional Annotation of DEGs

Experiment II:

Study the Expression of Genes induced
during Skinning Injury

Semiquantitative Reverse
Transcription PCR

Experiment III:

Quantitative Reverse
Transcription PCR

Using Susceptible and Resistant Cultivars of Sweetpotato to Study
the Effect of Curing on the Expression of Skinning-induced Genes
in LA 07-146 and LA 10-70 Cultivars of Sweetpotato

Isolation of Total RNA from LA 07-146 and LA 10-70

cDNA synthesis

QRT-PCR to Identify Genes/Transcripts
That Responsive to Curing

Figure 1.1 Research flowchart.

6

2 LITERATURE REVIEW
Sweetpotato is the seventh largest food crop, just after cassava, with an annual
production around 110 Mt and a cultivated area of 8.18 Mha (FAO 2013). It
produces stable crop yields under a wide range of environmental conditions and one
of the staple diets in many countries. Over 95% of the global sweetpotato crop is
produced in the developing countries, where it is the fifth most important crop on
fresh weight basis after rice, wheat, maize, and cassava (Plucknett 1991). Indonesia
is the forth world’s largest in sweetpotato production after China, Uganda and
Nigeria with the total production 2.39 Mt (FAO 2013). In Indonesia, sweetpotato is
utilized mainly as human food, 88% of total production in 1996 (World Bank 1998–
99 report summarized by CIP, 1999) although it could be a lower figure if it is true
as claimed by Jusuf (2002) that the crop’s use as food in recent years in Indonesia
has decreased but reliable statistics are lacking. Thus, varieties widely grown are
those preferred on the basis of traits related to eating quality such as taste and flavor
and root appearance rather than root yield (Jusuf 2002; Zuraida 2003). The most of
the popular local varieties of sweetpotato in Indonesia have colored flesh ranging
from cream to orange and at least in one case (Samarinda), purple (Zuraida 2003).

Skin Formation in Storage Roots and Wound Healing Processes
The storage roots of sweetpotato consist of the inner flesh, which is mainly
composed of parenchyma cells with starch granules (storage parenchyma), covered
by the skin. The skin is the secondary protective layer or periderm that consists of
three layers: phellem, phelloderm, and phellogen (Järvinen et al. 2011). The
phellogen originates the phelloderm toward the inside of the root and the phellem
toward the outside. The phellem is composed of several layers of cells devoid of
starch granules and positioned in a radial manner toward the phellogen. The
phelloderm is more difficult to identify as it resembles cortical cells and it usually
is distinguished by radial position of the cells in reference to the phellogen (Kono
and Mizoguchi 1982; Villavicencio et al. 2007; Firon et al. 2009).
The outer peridermal cells of sweetpotato become partly lignified during
growth and are progressively sloughed off. The phellogen layer, however, remains
active until harvest, and its activity caused the thickness of the periderm layer to
remain constant (Artschwager and Starrett 1931; Villavicencio et al. 2007; Firon
et al. 2009). The capacity to produce new periderm upon wounding is retained by
all parenchyma tissues in the tuber, but it is most pronounced close to the original,
native periderm (Artschwager 1927) (Fig. 2.1). In both wound and native periderm,
the phellem cells are identified by their rectangular shape and arrangement in
columnar rows and their walls autofluoresce under UV illumination (Ginzberg
2008). This columnar pattern is a result of their origin from periclinal division in
the phellogen cell layer (Artschwager 1924); the auto-fluorescence is due to the
presence of aromatic suberin polymers in their cell walls (Bernards and Lewis
1998).

7

Figure 2.1

Potato native and wound periderm. Cross sections of potato tuber
surface were stained with Safranin O/Fast Green and viewed with
light and UV microscopes to examine tissue morphology and auto
fluorescence of suberized cells, respectively. (a) Native periderm of
mature tuber with suberized phellem cells (the skin) and
parenchyma-like phelloderm, (b) Development of wound periderm
following removal of the skin. Wounding induced suberization of
the exposed tuber cells (1-3 d). On the third day a meristematic
phellogen that appears as multinuclear region developed below
these cells (Circled). Outward divisions of the phellogen produced
the characteristic suberized phellem layers (4-6d). The dark grains
are amyloplasts (Ginzberg, 2008).

The main problem in postharvest handling of sweetpotato roots is the loss of
the epidermis/skin from the surface of the root, referred to as “skinning”. Skinning
occurs when the superficial layers of the periderm separate from underlying tissue;
causing an increased rate of moisture loss, weight loss, shriveling of the root
surface, increase susceptibility to pathogen attack, and unattractive appearance. In
the United States, one of the most popular cultivars is ‘Beauregard’ because of its
smooth skin, color, and high yields. This cultivar, however is very prone to skin
loss during harvest and handling (Firon et al. 2009).
In sweetpotato storage roots, the process of wound-healing involves
desiccation of the surface cell layers, followed by lignifications of underlying cell
layers and finally the formation of a wound periderm by cell division (Artschwager

8

and Starrett 1931). At low humidity, poor wound healing is associated with a thick
desiccated layer and slow incomplete lignifications (van Oirschot et al. 2006).
Continuity of the lignified layer is vital for effective wound-healing, presumably to
act as an effective barrier to control fluxes of water, mineral nutrients and essential
gasses, and also defend against incorporation of toxic compounds, water loss, insect
predicament and pathogen invasion (van Oirschot et al. 2006; Ranathunge et al.
2011). A method for assessing efficiency of wound-healing based on van Oirschot
et al. (2002) assessing the continuity of lignified layers by phloroglucinol staining
(lignifications score: LS) was developed (van Oirschot et al. 2002; 2006), and has
been used as a tool for screening sweetpotato germplasms.
Villavicencio et al. (2007) suggested that because skin loss occurs due to
breakage of the cell walls of peridermal cells, lignin content and polygalacturonase
(PG) and pectin methylesterase (PME) activities could be part of the processes or
factor that lead to increased or decreased skin adhesion in sweetpotato roots. If this
is the case skin adhesion might correlate with these variables, making it possible to
use them as indicators of susceptibility to skinning.

Skinning Injury and Postharvest Loss
Physical Factors
Mechanical damage is the most important harvest factor, much of which is
sustained during harvest itself, and during transport and marketing. Harvesting in
the tropics is usually manual, employing a variety of tools such as digging sticks,
spades, hoes and knives. Sweetpotato roots are often cut, skinned and bruised by
the harvesting tools.
Physiological Factors
In sweetpotato, respiration and transpiration contribute to loss in weight and
alteration of internal and external appearance. Transpiration losses are due to`
evaporative loss of the cellular water caused by vapor pressure difference between
the root interior and the outside environment. In addition, wounding of sweetpotato
roots resulted in an increase in both the respiration rate and subsequent weight loss.
Furthermore, loss of moisture leads to a condition known as
‘pithiness‘ characterized by the formation of cavities within the tissues. Prolonged
moisture losses, as occur in tropical conditions may cause collapse of tissues starts
at the distal end of the roots. This is commonly found in small sized root which
could lead to total desiccation (Picha 1986).
Biological Factors
Pre-harvest and postharvest infections by pathogenic microorganisms (mostly
fungi and to a lesser extent bacteria) are serious causes of postharvest loss of
sweetpotato roots (Snowdon 1990). The relative importance of the major pathogens
can differ considerably between localities and with environmental conditions (Ray
and Byju 2003).

9

Strategies Adopted by Plants to Avoid Skinning Injury
Plants have evolved different mechanisms for protection against injury from
insect and micro-organism due to skinning. Structurally, plants have a polyester
coating composed of cutin and suberin (Kolattukudy 1980). This coating normally
isolates the plant tissues from competing organisms and plants are therefore
relatively immune from the presence of these competitors even on their surface.
However, if a break or wound occurs in this surface coating, then competing
organisms gain entrance into the plant’s tissues where they can cause injurious
damage to those tissues. Consequently, plants have developed a complex response
to wounding that dramatically alters the cellular physiology of plant tissues and
results in the production of defenses. These defenses are particularly protection
against microorganisms and are effective against small herbivores (Constabel et al.
2000).
A major phase of the wound-response is a generalized activation of plant
defenses. Because the majority of microbial infections occur in plants following a
wound, plants have developed a range of biochemical defenses to inhibit invading
pathogens and small herbivores (Zhou and Thornburg 1999). The accumulation of
phytoalexins after wounding has been a worldwide area for study. Phytoalexin, a
small molecular weight defensive compounds, has been shown to have a biological
activity against microorganisms or herbivores. For example, phenolic, terpenoid,
and alkaloid compounds are a major component of plant secondary metabolism
(Zhou and Thornburg 1999).
Plants protect themselves by physical barriers (such as wax coatings and
harden tissues), and chemical metabolites against damage from outside (Chen et al.
2005). Mechanical wounding and pathogen infection stimulate plants to produce
local and systemic signals, which in turn activate signal pathways within cells
(Bergey et al. 1999). Several compounds, including hydrogen peroxide, nitric
oxide, calcium, protein kinase, jasmonate, salicylic acid, and ethylene, were
involved in the signal pathways of defense systems (Delaney et al. 1994; Song et
al. 1995; O'Donnell et al. 1996; McConn et al. 1997; Zimmermann et al. 1997;
Foissner et al. 2000; Orozco-Cárdenas et al. 2001; Gould et al. 2002; Jih et al. 2003;
Deng et al. 2013).

Skinning Injury Induced Changes in Gene Expression
Sweetpotato storage roots are underground storage organs covered by skin or
periderm, a suberized layer that protects inner flesh from dehydration and
pathogens. Understanding the molecular processes associated with periderm
formation is of great importance for a better knowledge of this protective tissue and
for improving the storage life of storage roots. Moreover, understanding the
molecular mechanisms underlying skinning injury, attempts have turned toward the
isolation of genes regulated by skinning injury. This also permits insight into their
functions and the pathways that lead to their expression. Although several responses
of plants to skinning injury, including short term metabolic and physiological
changes may not require changes in gene expression, the majority are predicted to
rely on alterations in gene expression. The most important questions to be asked

10

with respect to skinning injury and other related biotic and abiotic stresses are: (1)
what genes are induced or repressed? (2) what is the function of the encoded gene
products? And (3) how are these genes regulated? (Bray 1993).
A common technique that has been used to identify and isolate
skinning/wound injury responsive has been the screening of cDNA library
constructed from poly(A)+ RNA from skinning injury plants. The encoded products
of the genes isolated by this technique are believed to play a role in a number of
processes including lignin and suberin biosynthesis pathways, protein fate, cell wall
modification, transcription and protein synthesis, and stress response and defense.
Genes Involved in Lignin and Suberin Biosynthesis Pathways
Suberin, a complex polymer, consists of aliphatic and aromatic domains
(Bernards 2002). The aliphatic suberin is a glycerol-based polyester of long and
very long chain of ω-hydroxyacids and fatty α, ω-diacids with small amounts of
esterified hydroxycinnamic acids, mainly ferulic acid. The aromatic suberin is a
lignin-like polymer mostly made of hydroxycinnamic acids. Regarding candidate
genes for aliphatic suberin pathway, two genes induced in potato skin are longchain acyl-CoA synthethase (LACS) apparently related to the activation of fatty
acids before elongation (Pollard et al. 2008) and a cytochrome P450 of the CYP94A
subfamily, whose putative orthologue in Nicotiana tabacum catalyses the oxidation
of fatty acids to α, ω-diacids (Le Bouquin et al. 2001). For aromatic suberin
pathway, it is worth to stress the presence of phenylalanine ammonia lyase (PAL)
and caffeic acid 3-O-methyl transferase (COMT) (Fig. 2.2). PAL encodes the
enzyme that catalyses the first step in the phenylpropanoid pathway (Kolattukudy
1981) whose enzymatic activity in periderm is concomitant with suberin deposition
(Bernards et al. 2000; Lulai 2008). COMT catalyzes the multi-step methylation
reactions of hydroxylated monomeric lignin precursors. It is believed to occupy a
pivotal role in the lignin biosynthesis pathway. A cDNA (TaCM) was identified
from wheat and expressed constitutively in stem, leaf, and root tissues (Ma and Xu
2008). The deduced amino acid sequence of TaCM showed a high degree of identity
with COMT from other plants, particularly in SAM binding motif and the residues
responsible for catalytic and substrate specificity (Ma and Xu 2008).
Genes Involved in Protein Fate
A group of protein encoded by the cDNA clones are involved in protein fate,
some of which are previously reported to be induced under various stress
treatments. Parvulin-type peptidyl-prolyl cis-trans isomerase (PPIase) is believed
to play a role in the folding of certain proteins by catalyzing the cis-trans
isomerization of X-Pro peptide bonds. Accumulation of PPIase mRNA was found
in plant responses to various environmental stress (Godoy et al. 2000; Reilly et al.
2007; Effendy et al. 2013). Gene of Soy max translationally controlled tumor
protein (SmTCTP) involved in protein fate (Gnanasekar et al. 2009). SmTCTP was
found to bind to native protein and protect them from thermal denaturation.
Overexpression of TCTP in bacterial cells can protect them from heat shockinduced cell death. This finding suggests that TCTP may belong to a novel small
molecular weight heat shock protein and can protect cellular proteins from heat
shock damage by acting as a molecular chaperone (Gnanasekar et al. 2009).

11

Figure 2.2 The monolignol biosynthesis pathway and typical lignin
distribution in monocot Switchgrass and dicot (Zhao and Dixon
2011)
Genes Involved in Cell Wall Modification
Changes in cell wall modifications are crucial during tuber periderm
differentiation as a result of cell extension and suberin deposition. At the subcellular
level, suberin is deposited inside the primary cell wall. Within subdomains of cell
walls, suberin occurs in three forms, depending on the developmental stage of the
suberized tissue (Ranathunge et al. 2011). Genes involved in cell wall growth and
remodeling such as pectin-glucuronyltransferase (Iwai et al. 2002; Soler et al.
2011), extensin (Cannon et al. 2008; Soler et al. 2011; Effendy et al. 2013;
Neubauer et al. 2013), xyloglucan endotransglucosylase/hydrolase (Fry 2004; Soler
et al. 2011) and -D-glucan exohydrolase (Hrmova and Fincher 2001) have been
found in the potato skin SSH library (Soler et al. 2011).
Genes Involved in Transcription and Protein Synthesis
Recent studies have revealed differentially expressed genes that involved in
transcription and protein synthesis (Zhou and Wu 2009; Gray et al. 2012).
Transcriptional and posttranscriptional regulations are important for gene
expression in eukaryotes. Transcription factors fulfill the work through transregulation to control plant responses to stress (Singh et al. 2002). The NAC protein
family is one of the largest families of plant specific transcription factors (Olsen et
al. 2005; Zheng et al. 2009). Genes from this family involved in various biological
processes including biotic and abiotic stress responses including drought and salt
stress (Zheng et al. 2009). Zinc finger proteins belong to the largest family of
regulatory transcription factors. They have very different structures and functions

12

for example RNA or DNA recognition, RNA packaging, transcriptional activation,
protein folding and assembly, and lipid binding (Laity et al. 2001; Zhou et al.
2009a; Zhou and Wu 2009).
Genes Involved in Stress Response and Defense
Periderm is a tissue that replaces the epidermis early in potato-tuber
development (Reeve et al. 1969). It was found that periderm is a tissue with high
exogenous and endogenous oxidative stress (Pla et al. 2000). Genes involved in
abiotic and biotic stress tolerance such as patatin-like phospholipase, catalase and
ascorbat peroxidase have been identified in both native and wound-healing
periderm (Chávez et al. 2005; Krits et al. 2007; Barel and Ginzberg 2008; Ginzberg
et al. 2009; Soler et al. 2011). Studies by Bernards (2002) showed that the
occurrence of stress proteins in periderm has been related with detoxification of the
reactive oxygen species (ROS). Furthermore, genes that regulate the protein redox
state, such as thioredoxin, are also up-regulated in periderm (Bernards 2002). Taken
together, the presence of genes involved in antioxidant activity and in redox
homeostasis suggests the importance of redox signaling (Buchanan and Balmer
2005; Foyer and Noctor 2005), which could have a main role in periderm.
Lectins have also been identified as defense-related proteins. Lectins are
carbohydrate-binding proteins, and commonly found in viruses, bacteria, plants,
and animals (Barondes 1