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Effects of oxylipin on pathogen
infection, physiological active
substances and aroma volatile synthesis
in grapes
January 2016
Wang Shanshan
Graduate School of Horticulture
CHIBA UNIVERSITY
(千葉大学学位申請論文)
ブドウの病原菌感染、生理活性物質お
よび香気成分合成に及ぼす
オキシリピンの影響
2016年1月
千葉大学大学院園芸研究科 環境園芸専攻生物資源科学コース
王珊珊
i
TABLE OF CONTENTS
List of abbreviations .................................................................................................... v
Chapter 1 General introduction and literature review ................................................. 1
1.1 General introduction ...................................................................................... 2
1.2 Literature review ............................................................................................ 7
1.2.1 Basic pathology of anthracnose disease .............................................. 7
1.2.2 Schematic of grape berry growth and development ........................... 8
1.2.3 Plant hormones and fungal defense .................................................. 11
1.2.3 Aroma volatiles biosynthestic pathways and enzymes ..................... 17
1.2.4 Aroma volatiles and fungal defense .................................................. 22
1.2.6 Retrospect of PDJ application ........................................................... 23
1.2.7 Retrospect of KODA application ...................................................... 23
Chapter 2 Jasmonate application influences endogenous abscisic acid, jasmonic acid
and aroma volatiles in grape berries infected by a pathogen (Glomerella cingulata) 25
2.1 Introduction .................................................................................................. 26
2.2 Materials and methods ................................................................................. 28
2.2.1 Plant material .................................................................................... 28
2.2.2 Fungal strain, culture, and inoculation .............................................. 29
2.2.3 Quantitative analysis of ABA and PA .............................................. 30
2.2.4 Jasmonic acid analysis ...................................................................... 30
ii
2.2.5 Volatile compound analysis .............................................................. 31
2.2.6 Measurement of O2− radical-scavenging activity ............................. 32
2.2.7 RNA extraction, cDNA synthesis, and quantitative real-Time
RT-PCR analysis ........................................................................................ 33
2.2.8 Statistical analysis ............................................................................. 35
2.3 Results .......................................................................................................... 36
2.3.1. Lesion diameter ................................................................................ 36
2.3.2. Endogenous abscisic acid (ABA), phaseic acid (PA) and expression
Of VvNCED1 And VvCYP707A1 .............................................................. 37
2.3.3 Endogenous jasmonic acid (JA), ethylene production, and the
changes in the O2-radical scavenging activities ......................................... 37
2.3.4 Aroma volatiles ................................................................................. 41
2.4 Discussion .................................................................................................... 45
Chapter 3 α-Ketol linolenic acid (KODA) application affects endogenous abscisic
acid, jasmonic acid, and aroma volatiles in grape berries infected by a pathogen
(Glomerella cingulata) .............................................................................................. 49
3.1 Introduction .................................................................................................. 50
3.2 Materials and methods ................................................................................. 53
3.2.1. Plant material ................................................................................... 53
3.2.2. Fungal strain, culture, and inoculation. ............................................ 54
3.2.3 Quantitative analysis of ABA and PA .............................................. 54
iii
3.2.4 Jasmonic acid analysis ...................................................................... 55
3.2.5. Volatile compound analysis ............................................................. 55
3.2.6 Measurement of O2− radical-scavenging activity ............................. 55
3.2.7 RNA extraction, cDNA synthesis, and quantitative real-Time
RT-PCR analysis ........................................................................................ 55
3.2.8 Statistical analysis ............................................................................. 57
3.3 Results .......................................................................................................... 58
3.3.1. Lesion diameter ................................................................................ 58
3.3.2. Endogenous abscisic acid (ABA), phaseic acid (PA) and expression
Of VvNCED1 And VvCYP707A1 .............................................................. 59
3.3.3 Endogenous jasmonic acid (JA), expression of VvLOX and VvAOS,
ethylene production, and the changes in the O2− radical scavenging
activities ..................................................................................................... 59
3.3.4 Aroma volatiles ................................................................................. 64
3.4 Discussion .................................................................................................... 66
Chapter 3 General conclusion ............................................................................. 71
Summary ............................................................................................................. 75
Acknowlegement ................................................................................................. 77
Referemces .......................................................................................................... 79
List of abbreviations
iv
LIST OF ABBREVIATIONS
GA gibberellic acid
ABA abscisic acid
PA phaseic acid
JA jasmonic acid
SA salicylic acid
G. cingulate Glomerella cingulata
ET ethylene
CK cytokinin
LOX lipoxygenase
MeJA Methyl Jasmonate
PDJ n-propyl dihydrojasmonate
KODA 9,10-ketol-octadecadienoic acid
Q-PCR quantative-PCR
AOS allene oxide synthase
AOC allene oxide cyclase
OPDA cis (+)-12-oxophytodienoic acid
◦C degree Celsius
List of abbreviations
v
µg microgram
µL microliter
µM micromolar
% percentage
DAFB days after full bloom
et al. et. alli (Latin), and others
GC-MS gas chromatography-mass spectrometry
g gram
h hour
HPLC high performance liquid chromatography
L liter
M Molar
mg milligram
mm millimeter
min minute
mL milliter
mM millimolar
m/s mass/charge ratio
nm nanometer
pH power of hydrogen ion
List of abbreviations
vi
RNA ribonucleic acid
rpm revolution per minute
sec second
SIM selected ion montoring
v/v volume by volume
w/v weight by volume
BHT butylated hydroxytoluene
KI Kovats Index
Chapter 1 General introduction and literature review
1
CHAPTER 1
GENERAL INTRODUCTION AND LITERATURE
REVIEW
Chapter 1 General introduction and literature review
2
1.1 GENERAL INTRODUCTION
Plant hormones act as a crucial role in controlling growth and development
of plants. The plant hormones such as auxin, cytokinin (CK), gibberellic acid (GA),
abscisic acid (ABA), jasmonic acid (JA), and ethylene (ET) can regulate cell division,
expansion and differentiation and so on (Tuteja and Sopory 2008). These effects can
be highly complex, with a single cell responding to multiple hormones and a single
hormone having differential effects on distinct tissues (Spartz and Gray, 2008).
Plants undergo continuous exposure to various biotic and abiotic stresses in
their natural environment. In response to microbial attack, plants stimulate a complex
series of responses that lead to the local and systemic induction of a broad spectrum
of antimicrobial defenses (Hammond-Kosack et al., 1996). The fungi Glomerella
cingulata has been associated with diseases in a wide range of host plants, and it can
infect fruits, flowers, leaves, and other non-lignified tissue (Weir et al., 2012). It is
well-known to cause anthracnose disease (ripe rot) on grapes and apples, which leads
to commercial losses annually (Southworth, 1981). Infections occurring before
veraison typically remain “dormant” until fruit begin to ripen in development of
grapes.
Recently, many progresses, including the cloning and characterization of
plant disease resistance genes that govern the recognition of specific pathogen strains,
Chapter 1 General introduction and literature review
3
have been made (Dangl and Jones, 2001; Staskawicz, 2001). The identification of
components are involved in the signal transduction pathways coupling pathogen
recognition to the activation of defense responses (Glazebrook, 2001; Feys and
Parker, 2000), and the demonstration that three endogenous plant signaling
molecules, salicylic acid (SA), JA, and ethylene, are involved in plant defense (Dong,
1998; Thomma, 2001). In addition, many other hormones, including ABA, GA, auxin,
and CK, have been shown to differentially affect Arabidopsis resistance against
biotroph and necrotroph by feeding into the SA-JA-ET cascades (Robert-Seilaniantz
et al., 2007, 2011).
Plants could have interaction with their environment by emitting volatile
organic compounds. Aroma volatile emissions influenced by pathogen inoculation
could be correlated with the ethylene production in Japanese apricot (Prunus mume
Sieb.) (Nimitkeatkai et al., 2011), and also varied by inoculation of fungus in
‘Mclntosh’ apple (Malus domestica Borkh.) (Vikram et al., 2004). From the aspect of
chemical characterization, volatiles can be classified as esters, alcohols, aldehydes,
ketones, lactones, and terpenoids (Hadi et al., 2013). Among these compounds,
C6-volatiles derived from the lipoxygenase (LOX) pathway, which including
C6-aldehydes and C6-alcohols act as a wound signal in plants, but result in a
moderate plant response relative to Methyl Jasmonate (MeJA) at both the
physiological and molecular level in Arabidopsis (ecotype Columbia) (Bate and
Chapter 1 General introduction and literature review
4
Rothstein, 1998). Many interactions of plants with pathogens also can depend on the
quantity and identity of terpenoids produced by plants (Bohlmann et al., 1998).
Jasmonic acid derivative, n-propyl dihydrojasmonate (PDJ) have been
applied for fruit thinning on Japanese pear ‘Hosui’ (Pyrus pyriforia Nakai), aroma
volatiles biosynthesis and accumulation of anthocyanidin in apple (Malus domestica
Borkh.), and decreased the lesion diameter on climacteric fruit Japanese apricot
(Prunus mume Sieb.) (Kondo and Mattheis, 2006; Kondo et al., 2000; Ohkawa et al.,
2006). However, there is not any cue on the relationship of PDJ treatment and fungus
defense in non-climacteric fruit grape. Moreover, complex crosstalk of plant
hormones involved in resistance system in grapes is also unclear.
α-Ketol linolenic acid (KODA): [9,10-ketol-octadecadienoic acid or
9-hydroxy-10-oxo-12(Z), 15(Z)-octadecadienoic acid] was identified as a
stress-induced substance in duckweed (Lemna paucicostata) (Yokoyama et al., 2000).
In oxylipin pathway, both KODA and JA are metabolized via LOX and allene oxide
synthase (AOS), known as AOS pathway (Yamaguchi et al., 2001; Creelman and
Mullet, 1997).
Chapter 1 General introduction and literature review
5
Fig. 1.1. The pathway for KODA and JA biosynthesis in higher plants (Kitticorn et
al., 2010)
(JA)
Chapter 1 General introduction and literature review
6
The objective of this study is to investigate the roles of PDJ and KODA on
resistance to fungus pathogen in grapes as follows:
1) Effects of PDJ and KODA application on lesion caused by fungus
inoculation.
2) Analysis of ABA, PA, JA, aroma volatiles and antioxidant activity after
inoculation and treatment.
3) Investigation of the interaction among JA, ABA, and aroma volatiles on
plant resistance to pathogen.
4) To detect the influences of PDJ and KODA treatments on the key genes
involved in biosynthetic pathways of plant hormones.
Chapter 1 General introduction and literature review
7
1.2 LITERATURE REVIEW
1.2.1 Basic pathology of anthracnose disease
Anthracnose disease, usually called ripe rot, is a disease that occurs on
ripening berries or near harvest (Christensen et al., 1890). The disease can be a
particularly devastating disease of muscadine grapes at warm and humid weather,
and its pathogen also causes fruit rots on series of other fruit and vegetable crops.
Circular, uniformly brown lesions develop on ripening fruit and eventually cover the
entire berry. Tiny black fruiting bodies form in the diseased tissue and exude masses
of salmon-colored to pink spores. Once the entire berry is rotted, it may drop to the
ground or remain on the vine as a mummy. Ripe rot is caused by the fungus
Glomerella cingulata (asexual stage: Colletotrichum gloeosporioides). This fungus
overwinters in fruit mummies and infected fruit stems. Spores (conidia) are released
in the spring and spread via splashing or blowing rain. While infections can occur at
any time, even when the fruit is still immature, decay does not begin until the fruit
ripens. Abundant conidia are produced on rotting fruit and spread to other ripe fruit.
Heavy losses can occur when frequent rains, coupled with warm temperatures, occur
during harvest. Although fungicides used early in the season may be effective for
limiting the damage caused with anthracnose. In view of characteristic of the
pathogen, condition in fruit ripening is usually favorable for ripe rot. Therefore,
Chapter 1 General introduction and literature review
8
application of fungicides should be deliberated.
1.2.2 Schematic of grape berry growth and development
Anthesis
Flower opening, commonly referred to as anthesis or bloom, occurs when
the calyptra detaches from the flower base and is shed, exposing the stamens and
pistil (Christensen and Andris, 1983). Anthesis normally occurs 6 to 8 weeks after
the commencement of shoot growth, depending on climatic conditions. Anthesis
proceeds rapidly when temperatures range between 29°C and 35°C.
Pollination and fertilization
Immediately after anthesis, the anthers split open and release their pollen
grains (Hardie et al., 1996). Grape varieties with hermaphroditic flowers are
considered self-pollinating, and the activity of insects or the presence of wind is
believed to be unnecessary for pollination. Pollination is complete once the pollen
has reached the stigma. If environmental conditions are favorable, the pollen grains
germinate and form pollen tubes. Fertilization occurs when sperm reaches and
impregnates the eggs within the embryo sacs. Under normal field conditions,
fertilization typically occurs two to three days after pollination. Temperature is an
important factor controlling germination and pollen tube growth. Optimum
temperatures for both germination and pollen tube growth range between 26.7°C and
Chapter 1 General introduction and literature review
9
32.2°C, and both processes are greatly reduced or even inhibited when temperatures
fall below 15.6°C or rise above 37.8°C.
Fruit set
In grapes, fruit set is defined as the stage when the berry diameter is
between 1⁄16 and 1⁄8 inch (1.6 and 3.2 mm) (Coombe, 1976; Lavee and Nir, 1986).
There are two other fruit set mechanisms, however, that allow seedless or seemingly
seedless berries to form. The first mechanism, parthenocarpy, is the only method by
which truly seedless berries are produced. The second mechanism, stenospermocarpy,
results in the formation of berries that appear to be seedless but are not. Several other
factors influence seed trace development, including the vine’s rootstock and year,
also climatic factors have a significant effect on fruit set.
Grape Berry Growth
Following fruit set, the grape flower ovary develops and advances through
three distinct stages of development during their growth (Harris et al., 1968; Pratt,
1971) (Fig. 1.2):
Stage I: the first phase of rapid berry growth. A period of rapid berry
growth begins cell division and enlargement immediately after bloom. Berry is firm,
while its color is green due to the presence of chlorophyll. The sugar concentrations
of the berry are low, while organic acids are high.
Chapter 1 General introduction and literature review
10
Fig. 1.2. Diagram to indicate berry and colour development measured at 10 day
intervals from just after flowering. The times when other components start to
accumulate, as well as the flow of xyleme and phloem juice in the berry, are
indicated (Kennedy, 2002).
Chapter 1 General introduction and literature review
11
Stage II: the lag phase of berry growth. Berry growth slows markedly
during the second period while the berries’ organic acid concentration reaches its
highest level. Berries are still firm, but begin to lose chlorophyll.
Stage III: the phase of rapid berry growth and fruit ripening. The
resumption of rapid berry growth and initiation of ripening commences with the
beginning of this stage (Coombe and Bishop, 1980; Uhlig and Clingeleffer, 1998).
The term berry softening (in French, veráison), which characterizes the initial stages
of color development, is commonly used to describe the striking changes in fruit
characteristics that mark the beginning of stage III. Berries soften and lose
chlorophyll, while red pigments begin to accumulate in the skin in colored varieties.
Sugar also begins to accumulate, and the concentration of organic acids declines.
Aroma and flavor components accumulate in the fruit. Berry growth during this stage
is limited to cell enlargement, and normally lasts between 6 and 8 weeks.
1.2.3 Plant hormones and fungal defense
Plant growth and response to environmental cues are largely governed by
plant hormones. The plant hormones ethylene, JA, and SA play a central role in the
regulation of plant immune responses. In addition, other plant hormones, such as
auxins, ABA, CK, GA, and brassinosteroids, that have been thoroughly described to
regulate plant development and growth, have recently emerged as key regulators of
Chapter 1 General introduction and literature review
12
plant immunity. Plant hormones interact in complex networks to balance the
response to developmental and environmental cues and thus limiting defense
associated fitness costs. The molecular mechanisms that govern these hormonal
networks are largely unknown. Moreover, hormone-signaling pathways are targeted
by pathogens to disturb and evade plant defense responses.
Jasmonic acid
JA is an important signalling molecule for the activation of defence in
response to wounding, herbivores and pathogen attack (Rosahl and Feussner, 2004).
It is synthesized from α-linolenic acid by enzymes of the lipoxygenase pathway
(Feussner and Wasternack, 2002). Linolenic acid liberated from chloroplast
membranes, putatively by phospho-/galactolipases, is oxygenated by
plastid-localized 13-lipoxygenases to 13-hydroperoxyoctadecatrienoic acid, which
serves as a substrate for a number of cytochrome P450 enzymes. One of these, allene
oxide synthase (AOS), catalyzes the conversion of 13-hydroperoxyoctadecatrienoic
acid to an unstable epoxide, allene oxide, which is subsequently cyclized by allene
oxide cyclase (AOC) to cis (+)-12-oxophytodienoic acid (OPDA).
OPDA is also found in a membrane-bound form as sn1-O-(12-
oxophytodienoyl)-sn2-O-(hexadecatrienoyl)-monogalactosyl-diglyceride (Stelmach
et al., 2001). Further enzymatic conversions take place in the peroxisome, to which
OPDA is translocated. OPDA reductase (OPR3) reduces the double bond of the
Chapter 1 General introduction and literature review
13
cyclopentenone, leading to the formation of 12-oxophytoenoic acid. In the
peroxisome, three rounds of beta-oxidation are required to synthesize JA. Then JA
can in turn be converted to the volatile ester, MeJA, by the enzyme jasmonic acid
carboxyl methyltransferase (Song et al., 2005). A new type of peroxisomal
acyl-coenzyme A synthetase was identified in A. thaliana which is capable of
catalyzing the synthesis of the CoA ester of OPDA, as well as of
3-oxopentenyl-cyclopentane-hexanoic acid (Schneider et al., 2005). The
wound-inducible acyl-CoA oxidase gene ACX1 from Arabidopsis was shown to be
required for JA-dependent responses (Cruz Castillo et al 2004). Recombinant ACX1A,
encoded by a gene identified from a JA-deficient tomato mutant, is indeed able to
catalyze the first step in the β-oxidative chain shortening of 12-oxophytoenoic acid
(Li et al., 2005). Multifunctional proteins which possess 2-trans-enoyl-CoA
hydratase and 3-hydroxy-acyl-CoA dehydrogenase activities, as well as a 3-ketoacyl-
CoA thiolase are responsible for the β-oxidation steps (Afitlhile et al., 2005). Finally,
a thioesterase is presumably involved in the release of jasmonic acid from its CoA
ester. Mutants defective specifically in the JA biosynthetic pathway were reported
for the AOS and OPR3 genes of Arabidopsis. The AOS mutants dde2
(delayed-dehiscence; von Malek et al., 2002) and aos (Park et al., 2002) were unable
to accumulate JA in response to wounding; however, only a little on aos mutants and
their response to microbial pathogens are available especially in fruits so far.
Chapter 1 General introduction and literature review
14
Ethylene
Introduction ET is a gaseous plant hormone that affects myriad
developmental processes and fitness responses, including germination, flower and
leaf senescence, fruit ripening, leaf abscission, root nodulation, programmed cell
death, and responsiveness to stress and pathogen attack (Bleecker and Kende, 2000;
Johnson and Ecker, 1998). Host-derived ET has been hypothesized to be an
important signal for disease development. Previous studies have shown a correlation
between the timing of increased ethylene evolution in response to pathogen
infections and the development of chlorotic and wilting symptoms in many plant
species (Goto et al., 1980; Pegg, 1981), including tomato (Gentile and Matta, 1975).
The accumulation and increasedactivities ofthe ET biosynthetic enzymes
1-aminocyclopropane-1-carboxylic acid (ACC) synthase and ACC oxidase have
been localized to chlorotic tissue directly surrounding primary lesions in tobacco
leaves in response to tobacco mosaic virus (De Laat and Van Loon, 1983) and
Phytophthora infestans (Spanu and Boller, 1989) infections. The localized activities
of ET biosynthetic enzymes in tissues surrounding primary lesions suggest that host
ET production may be a response to the cell death that occurs during primary lesion
formation. Bent et al. (1992) were the first to use a genetic approach to determine
whether ethylene perception plays a causal role in foliar disease development. This
finding of a critical function for the downstream ET signal transduction element
Chapter 1 General introduction and literature review
15
EIN2 in the regulation of disease development is not easily reconciled with the
relative lack of importance of ETR1, the ethylene receptor. He concluded that either
EIN2 is a critical component of additional pathogen response pathways or the ein2-1
allele is much stronger than is the etr1-3 allele in reducing ET sensitivity.
Salicylic acid
SA was identified as a crucial signaling molecule required for the
expression of plant defence responses. Transgenic Arabidopsis and tobacco plants
that cannot accumulate SA due to the expression of the bacterial NahG gene
encoding a salicylate hydroxylase, are hyper-susceptible to virulent pathogens,
unable to mount systemic acquired resistance, and impaired, although not completely
compromised, in race-cultivar-specific resistance (Delaney et al., 1994; Rairdan and
Delaney, 2002). SA is also required for the hypersensitive cell death response, which
is usually associated with resistance (Alvarez, 2000). Salicylic acid signalling
proceeds via the ankyrin repeat-containing protein NPR1, encoded by the NON
EXPRESSOR OF PR1 gene, which was identified in mutant screens of Arabidopsis
(Cao et al., 1994). NPR1 is present in the cytoplasm, presumably as an oligomer, and
responds to pathogen-induced alterations in the cellular redox state by translocating
to the nucleus in its monomeric form (Fobert and Despres, 2005). In Arabidopsis,
pathogen-induced salicylic acid accumulation requires de novo biosynthesis via the
isochorismate pathway, since mutants compromised in SA accumulation carry a
Chapter 1 General introduction and literature review
16
defective gene for isochorismate synthase (Wildermuth et al., 2001). On the other
hand, labelling studies in tobacco revealed that SA is also produced via the
phenylpropanoid pathway from phenylalanine, cinnamic acid, and benzoic acid, or
conjugates thereof (Ribnicky et al., 1998). In potato, these compounds were also
shown to be precursors for salicylic acid synthesis in untreated plants; however, after
elicitor treatment, SA accumulated that was not derived from phenylalanine (Coquoz
et al., 1998), suggesting that here, as in Arabidopsis, pathogen or elicitor-induced
accumulation of SA involves synthesis via an alternative, possibly the isochorismate
pathway.
Abscisic acid
ABA concentrations in grape berries increase just before véraison;
therefore, ABA may be a signal of ripening in grape berries (Coombe and Hale,
1973). ABA is involved in the regulation of many aspects of plant growth and
development including seed germination, embryo maturation, leaf senescence,
stomatal aperture and adaptation to environmental stresses (Wasilewskaa et al. 2008).
In ABA biosynthesis in higher plants, the 9-cis-epoxycarotenoid dioxygenase
(NCED) is a key enzyme that cleaves 11, 12 double bonds of C40 carotenoids and
produces xanthoxin (Qin and Zeevaart, 1999). Subsequently, ABA is primarily
catabolized to form 8′-hydroxy ABA by hydrox- ylation with ABA 8′-hydroxylase,
and then isomerizes to phaseic acid (PA) (Nambara and Marion-Poll, 2005).
Chapter 1 General introduction and literature review
17
Several recent papers have reported that ABA plays important roles in
plant defence responses (Adie et al. 2007; de Torres-Zabala et al. 2007; Mauch-Mani
and Mauch 2005; Mohr and Cahill 2007). However, the role of ABA in plant defence
appears to be more complex, and vary among different types of plant-pathogen
interactions. Yasuda et al. (2008) reported that ABA treatment suppressed SAR
induction indicating that there is an antagonistic interaction between SAR and ABA
signaling in Arabidopsis, however, the role of ABA as a positive regulator of defence
has also been reported (Mauch-Mani and Mauch 2005). ABA activates stomatal
closure that acts as a barrier against bacterial infection (Melotto et al. 2006). These
results demonstrate that ABA is not a positive regulator of plant defence against all
necrotrophs and its role depends on individual plant pathogen interactions.
1.2.3 Aroma volatiles biosynthestic Pathways and enzymes
Plants are able to emit volatile organic compounds and the content and
composition of these molecules show both genotypic variation and phenotypic
plasticity (Maffei, 2010). Various types of fresh fruits produce distinct volatile
profiles. Fruit volatile compounds are mainly comprised of diverse classes of
chemicals, including esters, alcohols, aldehydes, ketones, lactones, and terpenoids
(Goff and Klee, 2006). Grape (Vitis vinifera L.) volatiles include a great number of
compounds, among which monoterpenes, C13-norisoprenoids, alcohols, esters and
Chapter 1 General introduction and literature review
18
carbonyls are found (Dieguez et al., 2003)
Since volatiles are comprised of at least five chemical classes, there are
several pathways involved in volatile biosynthesis. Volatiles important for aroma and
flavor are biosynthesized from membrane lipids, amino acids and carbohydrates
(Sanz et al.,1997).
Fatty acids pathway
Fatty acids are major precursors of aroma volatiles in most fruit (Sanz et
al.,1997). Fatty acid-derived straight-chain alcohols, aldehydes, ketones, acids, esters
and lactones ranging from C1 to C20 are important character-impact aroma
compounds that are responsible for fresh fruit flavors with high concentrations, and
are basically formed by three processes: α-oxidation, β-oxidation and the
lipoxygenase (LOX) pathway (Schwab and Schreier, 2002). Aroma volatiles in intact
fruit are formed via the β-oxidation biosynthetic pathway, whereas when fruit tissue
is disrupted, volatiles are formed via the LOX pathway (Schreier, 1984). Many of the
aliphatic esters, alcohols, acids, and carbonyls found in fruits are derived from the
oxidative degradation of linoleic and linolenic acids (Reineccius, 2006). In addition,
some of the volatile compounds derived from enzyme-catalyzed oxidative
breakdown of unsaturated fatty acids may also be produce by autoxidation
(Chan, 1987). Autoxidation of linoleic acid produces 9,13-hydroperoxides, whereas
linolenic acid also produces 12,16-hydroperoxides (Berger, 2007). Hexanal and
Chapter 1 General introduction and literature review
19
2,4-decadienal are the primary oxidation products of linoleic acid, while autoxidation
of linolenic acid produces 2,4-heptadienal as the major product. Further autoxidation
of these aldehydes leads to the formation of other volatile products (Chan, 1987).
Lipoxygenase (LOX)
The metabolism of polyunsaturated fatty acids, via the first LOX-catalyzed
step and the subsequent reactions, is commonly known as the LOX pathway.
Saturated and unsaturated volatile C6 and C9 aldehydes and alcohols are important
contributors to the characteristic flavors of fruits, vegetables and green leaves. The
short-chain aldehydes and alcohols are produced by plants in response to wounding
and play an important role in the plants defense strategies and pest resistance (Matsui,
2006; Stumpe and Feussner, 2006). At least four enzymes are involved in the
biosynthetic pathway leading to their formation: LOX, hydroperoxide lyase (HPL),
3(Z), 2(E)-enal isomerase and ADH. When fruit are homogenised, linoleic and
linolenic acid are oxidised to various C6 and C9 aldehydes (Lea, 1995). In intact fruit,
enzymes in the LOX pathway and their substrates have different subcellular
locations, preventing formation of volatile compounds (Chan, 1987). During ripening,
cell walls and membranes may become more permeable, allowing the LOX pathway
to become active without tissue disruption. The LOX biosynthetic pathway has the
potential to provide substrates for ester production (De Pooter et al., 1983). If the
LOX biosynthetic pathway were active during ripening, it would act as an alternative
Chapter 1 General introduction and literature review
20
to β-oxidation of fatty acids. As shown in Fig 1.3, volatile fatty acid derivatives such
as trans-2-hexenal, cis-3-hexenol and MeJA are derived from C18 unsaturated fatty
acids including linoleic acid or linolenic acid, which undergo dioxygenation in a
reaction catalyzed by LOX (Feussner and Wasternack, 2002). These enzymes can
catalyze the oxygenation of polyenoic fatty acids at C9 or C13 positions yielding two
groups of compounds, the 9-hydroperoxy and the 13-hydroperoxy derivatives of
polyenoic fatty acids. These derivatives can be further metabolized by an array of
enzymes, including AOS and HPL, which represent two branches of the LOX
pathway yielding volatile compounds. In the AOS branch of the LOX pathway,
13-hydroxyperoxy linolenic acid is converted to 12,13-epoxyoctadecatrienoic acid
by AOS. A series of subsequent enzymatic reactions leads to the formation of JA,
which can in turn be converted to the volatile ester, MeJA, by the enzyme jasmonic
acid carboxyl methyltransferase (Song et al., 2005). In the HPL branch of the LOX
pathway, the oxidative cleavage of hydroperoxy fatty acids through the action of
HPL leads to the formation of short chain C6 or C9 volatile aldehydes (e.g., 3-hexenal
or 3,6-nonadienal) and the corresponding C12 or C9 ω-fatty acids (e.g.,
12-oxo-dodecenoic acid or 9-oxononanoic acid). HPL C6 aldehyde products can be
further converted to their isomers by spontaneous rearrangement by alkenal
isomerases, or reduced to alcohols by the action of ADH (Akacha et al., 2005).
Chapter 1 General introduction and literature review
21
Fig 1.3. Linolenic acid-derived flavor molecules. AAT, alcohol acyl CoA transferase;
ADH, alcohol dehydrogenase; AER, alkenal oxidoreductase; AOC, allene oxide
cyclase; AOS, allene oxide synthase; HPL, hydroperoxide lyase; JMT, jasmonate
methyltransferase; LOX, lipoxygenase; OPR, 12-oxo-phytodienoic acid reductase;
3Z,2E-EI, 3Z,2E-enal isomerase (El Hadi et al., 2013).
Chapter 1 General introduction and literature review
22
1.2.4 Aroma volatiles and fungal defense
C6 compounds and fungal defense
In aroma volatiles, green leaves volatiles consist of a family of C6
compounds, including aldehydes, alcohols and esters, originate in the HPL branch of
the oxylipin pathway (Matsui, 2006). They are almost ubiquitously made by green
plants and increased release can be caused by herbivores (Fall et al., 1999) or
pathogens (Heden et al., 2003). Exogenous application of E-2-hexenal or
Z-3-hexenal to Arabidopsis plants induces the expression of VSP1 and AOS, showed
that C6 compounds may activating the accumulation of JA (Kishimoto et al., 2006).
Terpenoids and fungal defense
Recent work has shown that the octadecanoid molecule, methyl
jasmonate (MeJA), plays a substantial role in the induction of TPS genes
located terpenoid synthases and in defense-related changes in terpenoid
biochemistry and anatomy in spruce (Franceschi et al., 2002; Fa’ldt et al.,
2003; Martin et al., 2002). For example, while sapling trees emitted mainly
monoterpene hydrocarbons on a constitutive basis, the MeJA-induced tissues
released large amounts of oxygenated monoterpenes, e.g., linalool, and
sesquiterpenes, e.g., farnesene and bisabolene, which implied the interaction
of terpenoid and JA pathway (Martin et al., 2003).
Chapter 1 General introduction and literature review
23
1.2.6 Retrospect of PDJ application
Over decade, the synthetic analogue of MeJA: n-propyl dihydrojasmonate
(PDJ) were widely applied in many aspects on growth and development of fruits,
such as, thinning effect on Japanese pear (Pyrus pyrifolia var. culta. ) (Ohkawa et al.,
2006), promoting ripening by inducing ACC synthase gene ACS1 gene in ethylene
pathway (Kondo et al., 2007), increasing the production of esters (butyl propanoate,
butyl butyrate, and propyl butyrate) and of anthocyanin in apple (Malus sylvestris L.
Mill. var. domestica Borkh. Mansf.) by treating at pre-climacteric stage (Kondo and
Mattheis, 2006), decreasing the injury of pathogen Colletotrichum gloeosporioides in
climacteric fruit Japanese apricot (Prunus mume Sieb.), regulating the development
and growth of peach (Prunus persica L. Batsch) (Ziosi et al., 2008).
1.2.7 Retrospect of KODA application
α-Ketol linolenic acid (KODA): [9,10-ketol-octadecadienoic acid or
9-hydroxy-10-oxo-12(Z), 15(Z)-octadecadienoic acid] was identified as a
stress-induced substance in duckweed (Lemna paucicostata) (Yokoyama et al., 2000).
KODA is formed from 9-hydroperoxy-10(E), 12(Z), 15(Z)-octadecatrienoic acid
(9-HPOT) generated from linolenic acid via 9-lipoxygenase (9-LOX) (Vick and
Zimmerman 1987). KODA reacted as a flower-inducing compound in violet
(Pharbitis nil), carnation (Dianthus caryophyllus L.), and apple (Malus domestica
Chapter 1 General introduction and literature review
24
Borkh.) (Suzuki et al., 2003; Yokoyama et al., 2005; Kittikorn et al., 2010). In the
interaction with JA in biosynthesis pathway, KODA are assumed to be involved with
resistance against invasion of pathogen.
Chapter 2 Jasmonate application influences endogenous abscisic acid, jasmonic acid and aroma
volatiles in grapes infected by a pathogen (Glomerella cingulata)
25
CHAPTER 2
JASMONATE APPLICATION INFLUENCES
ENDOGENOUS ABSCISIC ACID, JASMONIC ACID,
AND AROMA VOLATILES IN GRAPES INFECTED BY
A PATHOGEN (GLOMERELLA CINGULATA)
Chapter 2 Jasmonate application influences endogenous abscisic acid, jasmonic acid and aroma
volatiles in grapes infected by a pathogen (Glomerella cingulata)
26
2.1 INTRODUCTION
Fungal disease spoils the quality of fruits and vegetables. G. cingulata is a
plant pathogenic fungus that causes disease in different hosts and anthracnose in
many fruit and vegetable species. Some phytohormones and secondary metabolites
are induced as responses to prevent development of the pathogen. For example, JA
levels rise steeply against damage caused by environmental stress, and trigger the
formation of many different kinds of plant defenses (Taiz et al., 2002). It has been
shown that jasmonates, especially JA and its methyl ester (MeJA) mediate resistance
to insects and disease (Creelman et al., 1997). A previous report (Nimitkeatkai et al.,
2011) showed that application of PDJ, which is a synthetic analog of JA, influenced
ethylene production and aroma volatiles, a response mediated by endogenous JA
levels in a Japanese apricot (Prunus mume Sieb.) infected by a pathogen. Japanese
apricot is a climacteric fruit, and ET is closely associated with environmental stresses
(Abeles et al., 1992). However, the relationship between JA and ethylene in grapes, a
non-climacteric fruit, when infected by fungus is unclear.
ABA concentrations in grape berries increase just before véraison;
therefore, ABA may be a signal of ripening in grape berries (Coombe et al., 1973).
ABA triggers responses (stomatal close, induction, etc.) against environmental stress
such as drought, high salinity, and herbicide toxicity (Cho et al., 2012; Grossmann et
Chapter 2 Jasmonate application influences endogenous abscisic acid, jasmonic acid and aroma
volatiles in grapes infected by a pathogen (Glomerella cingulata)
27
al., 1996). In ABA biosynthesis in higher plants, NCED is a key enzyme that cleaves
11, 12 double bonds of C40 carotenoids and produces xanthoxin (Qin et al., 1999).
Subsequently, ABA is primarily catabolized to form 8′-hydroxy ABA by
hydroxylation with ABA 8′-hydroxylase, and then isomerizes to PA (Nambara et al.,
2005). ABA can also prominently regulate pathogen defense responses
(Vleesschauwer et al., 2010). Therefore, ABA and JA may interact in grapes in
response to environmental stress.
In this study, the effects of PDJ application on lesion diameter, endogenous
JA, ABA, aroma volatiles, and antioxidant activity were investigated in grapes
infected by G. cingulata). In addition, changes in the expression of VvNCED1 and
ABA VvCYP707A1 genes in grapes were analyzed.
Chapter 2 Jasmonate application influences endogenous abscisic acid, jasmonic acid and aroma
volatiles in grapes infected by a pathogen (Glomerella cingulata)
28
2.2 MATERIALS AND METHODS
2.2.1 Plant material
Three 7-year-old ‘Kyoho’ grapevines [Vitis labrusca × V. vinifera] grafted
onto ‘Teleki-kober 5BB’ rootstocks [V. berlandieri × V.riparia hibrids] were
harvested at the véraison stage (50 days after full bloom [DAFB]). The vines had
been growing in an open field at Chiba University, located at 35 °N Lat, 140 °E
Long, and at an altitude of 37 m. Two times of 25ppm Gibberellin (GA) treatment
were applied for the development and seedless of grape berries (First time: at full
blood date; Second time: 10 days after full bloom). Berries of similar size and color
were selected and rinsed with tap water, then sterilized by 0.5% sodium hypochlorite
for 3 min and washed with distilled water prior to treatment. The fruits were
randomly divided into three groups of 800 berries. In the first and second groups, the
fruits were dipped into distilled water containing 0.1% (v/v) surfactant Approach BI
(50% polyoxyethylene hexitan fatty acid ester; Kao, Osaka, Japan) for 5 min. In the
third group, the fruits were dipped in 0.4 mM PDJ solution (Nippon Zeon Co., Tokyo,
Japan), containing 0.1% (v/v) Approach BI. All berries were then air-dried and
wounded with the needle of a syringe to a depth of 5 mm. Fruits from the second and
third groups were inoculated with G. cingulata.
Chapter 2 Jasmonate application influences endogenous abscisic acid, jasmonic acid and aroma
volatiles in grapes infected by a pathogen (Glomerella cingulata)
29
2.2.2 Fungal strain, culture, and inoculation
The G. cingulate (MAFF, 239927) used in this study as the anthracnose
pathogen was incubated on potato dextrose agar at 25°C for 7 days. The conidia were
collected with distilled water after the surface of the medium was scraped. Density of
the conidia suspension was adjusted to 106 conidia/mL with distilled water using a
hemosytometre (Thoma [EKDS Co., Tokyo, Japan], depth 1/10 mm, No. D7092,
1/400 sqmm).
The suspension with spores was dripped onto the wounded surfaces of the
fruit in the second and third groups, hereafter referred to as PDJ− inoc+ and PDJ+
inoc+, respectively. A suspension without spores was placed on the wounds of the
control fruit in the first group, hereafter referred to as control.
All fruit from the three groups were placed in a controlled room at 25 °C
and 95% relative humidity. Two hundred berries per treatment group (three
replications of 66-67 berries) were sampled at 3, 6, 9, and 12 days after treatment.
Before treatment (day 0), two hundred berries (three replications of 66-67 berries)
were collected immediately after harvest. After lesion diameter and ethylene
production were measured, the skin was sampled and frozen in liquid N2. The
samples were stored at −80°C.
Chapter 2 Jasmonate application influences endogenous abscisic acid, jasmonic acid and aroma
volatiles in grapes infected by a pathogen (Glomerella cingulata)
30
2.2.3 Quantitative analysis of ABA and PA
The frozen skin samples [1g fresh weight (FW); three replications] were
homogenized in 20 mL cold 80% (v/v) methanol including 0.5 g
polyvinylpyrrolidone with 0.2 µg ABA-d6 and PA-d3 as an internal standard. The
extraction and analysis of ABA and PA in samples were performed as previously
described by Kondo et al. (2012). The methyl ester of ABA and PA was analyzed by
gas chromatography-mass spectrometry-selected ion monitoring (GC-MS-SIM;
model QP5000; Shimadzu, Kyoto, Japan). The column temperature was a step
gradient of 60°C for 2 min, and thereafter was raised to 270°C at 10°C min−1; it was
held at 270°C for 35 min. The ions were measured as ABA-d0 methyl ester/ABA-d6
methyl ester at m/z 190, 260, 194, and 264. For PA, the ions were measured as
follows: PA-d0 methyl ester/PA-d3 methyl ester at m/z 276, 294, 279, and 297. ABA
concentration was determined from the ratio of peak areas for m/z 190 (d0)/194 (d6).
The PA concentration was calculated from the ratio of peak areas for m/z 276
(d0)/279(d3).
2.2.4 Jasmonic acid analysis
Extraction and analysis of JA were performed according to the procedure
described in a previous paper (Kondo et al., 2005) with GC-MS-SIM. One gram of
fruit peel was homogenized with 100 µL of ((±)- 2-(2, 3-2H2) JA (100 mg L-1)) as an
Chapter 2 Jasmonate application influences endogenous abscisic acid, jasmonic acid and aroma
volatiles in grapes infected by a pathogen (Glomerella cingulata)
31
internal standard in 10 mL of saturated NaCl solution and 20 mL of diethyl ether
containing 0.005% butylated hydroxytoluene (BHT) as an antioxidant. The aqueous
layer was extracted 3 times with 20 mL of diethyl ether containing 0.005% BHT.
The pooled ether extract was dried under warm air, then the residue was dissolved in
200 µL of chloroform/isopropylethylamine, 1:1 (v/v), and derivatized at 50°C for 60
min with pentafluorobenzyl bromide. The derivatization mixture were then dried
under N2. The residue was dissolved in 2mL n-hexane and added onto a silica gel
column. The sample was eluted with 7mL n-hexane/diethyl ether (v/v=2:1) then
dried at 50°C under N2. After dissolved in 1mL methanol, sample was filtrated
through Millipore. The ions were measured as m/z 392, 390, 211, and 209. The
concentration of JA in the original extract was determined from the ratio of peak
areas for m/z 209 (2H0)/211 (2H2).
2.2.5 Volatile compound analysis
0.1 g of grape peel was put in a 4-mL vial to which was added 1 µL of
1000-ppm cyclohexanol. The sample was stirred for 50 min at 25°C, and the
equilibrium of headspace volatile compounds between the grape matrix and the
headspace was accelerated. Volatile compounds extraction was then performed by
injecting a 10-µM polydimethylsiloxane SPME fiber (Supelco, Bellefonte, PA, USA)
into the vial and exposing it to the headspace for 5 min at 25°C. After extraction,
Chapter 2 Jasmonate application influences endogenous abscisic acid, jasmonic acid and aroma
volatiles in grapes infected by a pathogen (Glomerella cingulata)
32
samples were directly desorbed into the injection port of the GC, which was at 230°C.
The volatile compounds were analyzed with a Shimadzu GC-MS QP2010 (Shimadzu
Co., Ltd., Kyoto, Japan). Volatile compounds were separated using a capillary
column (DB-WAX; 60 m × 0.25 mm ID × 0.25-µM film thickness; Agilent, Santa
Clara, CA, USA), with a helium gas linear flow rate of 30 cm/min. The temperature
program was isothermal at 40°C for 7 min, then was raised 5°C/min to 220°C, and
finally raised 20°C/min to 245°C, where it was maintained for 15 min. The GC-MS
transfer line temperature was at 200°C. The mass spectrometry (MS) operated in
electron impact mode with electron impact energy of 70 eV, and collected data at a
velocity of 0.5 scans/s over a range of m/z 50–350. N-Alkanes were analyzed under
the same conditions to calculate the Kovats Index (KI) for volatile compounds. The
compounds were identified by comparison with commercial reference compounds.
These were provided by comparison of KI with those described in the literature, and
by comparison of their MS data with those contained in the National Institute of
Standards and Technology (NIST, 21 and 28).
2.2.6 Measurement of O2− radical-scavenging activity
The analyses of O2− radical-scavenging activity were performed according
to the method described in Kondo et al. (2005). Skin samples of 1g (three
replications) were homogenized in 10 mL 13.7 mol/L ethanol and filtered. The
Chapter 2 Jasmonate application influences endogenous abscisic acid, jasmonic acid and aroma
volatiles in grapes infected by a pathogen (Glomerella cingulata)
33
Potassium phosphate-borax-EDTA buffer (pH 8.2), 0.5mM Hypoxantin solution,
10mM Hydroxylammonium Chloride (Wako, Co., Ltd., Japan) and distill water was
mixed with sample then Xantineoxidase was added and incubated at 37°C for 30 min
in water bath and constantly shaking. A mixed solution with 30 mM of
N-(1-Naphthyl)ethylenediamine dihydrochloride, 3mM of Sulfanilic acid and 25%
Acetic acid then keep at room temperature for 30 min. The analysis with
spectrophotometer was at 550nm.
2.2.7 RNA extraction, cDNA synthesis, and quantitative real-time RT-PCR
Total RNA extraction from the skin (1 g FW; three replications) was
performed as previously reported by Kondo et al. (2012). The sample was pulverized
then incubated in 65°C water bath for 10 min with 200mg polyvinyl polypyrrolidine,
200 µL β-Mercaptoethanol and 10 mL extraction solution contained 2% CTAB
(Cetyltrimethylammonium Bromide). The mixture was extracted 2 times by the
equal volume of Chloroform: Isoamyl alcohol (24:1), and centrifuged at 9000×g, 4°C
for 10 min. The upper phase was transferred to a new tube then 10% volume 3M
NaOAc and 60% volume of isopropanol was added and kept at -80°C for 30 min.
After centrifuge at 3500×g, 4°C for 30 min and remove the solution, the pellet was
kept and rinsed with 70% ethanol then air-dried. 1 mL of 1% TE was used to
dissolve the pellet then transferred to a 2 mL tube. After add 120 µL of 10 M LiCl,
Chapter 2 Jasmonate application influences endogenous abscisic acid, jasmonic acid and aroma
volatiles in grapes infected by a pathogen (Glomerella cingulata)
34
the tube was kept at 4°C more than 2 hours. Centrifuge at 20000×g, 4°C for 30 min
and remove the supernatant then washed by 160 µL of 70% ethanol. After the sample
was dried then dissolved with 30 µL hyperpure water. CcDNA synthesis was carried
out according to the method described in Kondo et al. (2014) after the measurement
of RNA concentration and electrophoresis. Gene-specific primers for each gene
(Table 2.1) were used for RT-PCR. Primers for the amplification of the
VvCYP707A1 gene were based on a previous report (Kondo et al. 2014). The
nucleotide sequence of the fragment amplified with these primers was confirmed by
sequencing. Primers for the VvNCED1 gene were constructed based on the sequences
reported by Sun et al. (2010). The relative expression level of each gene was
determined by a relative standard curve method. The expression level was
normalized to that of the VvUbiquitin gene (Fujita et al. 2007).
Table 2.1. Primers used for real-time RT-PCR.
Gene Forward/reverse primer (5′ – 3′ ) Reference
VvNCED1 (F) GGTGGTGAGCCTCTGTTCCT Sun et al. (2010)
(R) CTGTAAATTCGTGGCGTTCACT
VvCYP707A1 (F) GAAACATTCACCACAGTCCAGA Kondo et al. (2014)
(R) AGCAAAAGGGCCATACTGAATA
VvUbiquitin (F) TCTGAGGCTTCGTGGTGGTA Fujita et al. (2007)
(R) AGGCGTGCATAACATTTGCG
Chapter 2 Jasmonate application influences endogenous abscisic acid, jasmonic acid and aroma
volatiles in grapes infected by a pathogen (Glomerella cingulata)
35
2.2.8 Statistical analysis
The data was presented as the mean values of the three replications ± the
standard error (SE), and separated by Fisher’s least significant difference (P≤0.05)
using the SAS analysis of variance procedure (version 8.2, SAS Institute, Cary, NC,
USA). The lesion diameter in Fig. 2.1 was evaluated using the Tukey-Kramer test at
each date.
Chapter 2 Jasmonate application influences endogenous abscisic acid, jasmonic acid and aroma
volatiles in grapes infected by a pathogen (Glomerella cingulata)
36
2.3 RESULTS
2.3.1. Lesion diameter
The diameters of the lesions infected by G. cingulata in the PDJ− inoc+ and
PDJ+ inoc+ samples are shown in Figure 1. Lesion diameters in PDJ+ inoc+ samples
were significantly smaller than those in PDJ− inoc+ at 9 and 12 days after treatment.
Fig. 2.1. Lesion diameter in ‘Kyoho’ grape berries after pathogen inoculation and
PDJ application. Values are from three replications of 50 fruits. Asterisks indicate
significant differences on each day after treatment (**, P < 0.01).
0
5
10
15
20
25
0 3 6 9 12 15
PDJ+ inoc+
PDJ- inoc+
Days after inoculation
Leis
ion
diam
eter
(mm
)
**
ns
**
ns
PDJ+ inoc+
PDJ− inoc+
Chapter 2 Jasmonate application influences endogenous abscisic acid, jasmonic acid and aroma
volatiles in grapes infected by a pathogen (Glomerella cingulata)
37
2.3.2. Endogenous abscisic acid (ABA), phaseic acid (PA) and expression of
VvNCED1 and VvCYP707A1
ABA concentrations in inoculated fruit increased at 3 days after treatment
(Fig. 2.2). In addition, the ABA concentrations in PDJ+ inoc+ were higher than those
in PDJ− inoc+. PA concentrations were highest in PDJ+ inoc+, followed by PDJ− inoc+;
lowest were in the untreated control. The expression levels of VvNCED1 in PDJ+
inoc+ were highest 6 and 12 days after inoculation, followed by PDJ− inoc+ and the
untreated control (Fig. 2.3). The expressions of VvCYP707A showed a similar
tendency with that of VvNCED1.
2.3.3 Endogenous jasmonic acid (JA), ethylene production, and the changes in
the O2-radical scavenging activities
In general, JA concentrations in PDJ+ inoc+ were higher than those in the
other two treatments (Fig. 2.4). The JA concentrations in PDJ+ inoc+ were highest at
9 and 12 days after treatment. There were no significant differences in ethylene
production between the three treatments through measured data (not shown). EC50
value was defined as the concentration of substrate that caused a 50% loss in O2−
scavenging activities. Low EC50 values show high antioxidant activity (Fig. 5). The
values of EC50 in PDJ+ inoc+ were lowest at 3 and 6 days after treatment.
Chapter 2 Jasmonate application influences endogenous abscisic acid, jasmonic acid and aroma
volatiles in grapes infected by a pathogen (Glomerella cingulata)
38
Fig. 2.2. Endogenous (A) ABA and (B) PA concentrations in ‘Kyoho’ grape skins
after pathogen inoculation and PDJ application. Data are the means ± SE of three
replications.
0
0.5
1
1.5
2
2.5
3
AB
A (µ
mol
/kg)
Control
PDJ-inoc+
PDJ+inoc+
LSD0.05 = 0.12 (A)
PDJ- inoc+
PDJ+ inoc+
Control
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
0 3 6 9 12 15
PA (
µmol
/kg)
Days after incoculation
LSD0.05 = 0.08
(B)
Chapter 2 Jasmonate application influences endogenous abscisic acid, jasmonic acid and aroma
volatiles in grapes infected by a pathogen (Glomerella cingulata)
39
Fig. 2.3. Quantitative real time RT-PCR analysis of (A) VvNCED1 and (B)
VvCYP707A1 in ‘Kyoho’ grape skins after pathogen inoculation and PDJ application.
Data are the means ± SE of three replications.
0
1
2
3
4
5
6
7
0 6 12 Days after inoculation
Rel
ativ
e Vv
CYP
'07A
1 ex
pres
sion
LSD0.05 = 0.61(B)
0
0.5
1
1.5
2
2.5 Control
PDJ-inoc+
PDJ+inoc+
LSD0.05 = 0.12
Rel
ativ
e vv
NC
ED1
expr
essi
on (A)
PDJ+ inoc+
Control
PDJ− inoc+
Chapter 2 Jasmonate application influences endogenous abscisic acid, jasmonic acid and aroma
volatiles in grapes infected by a pathogen (Glomerella cingulata)
40
Fig. 2.4. Endogenous JA concentrations in ‘Kyoho’ grape skins after pathogen
inoculation and PDJ application. Data are the means ± SE of three replications.
0
20
40
60
80
100
120
0 3 6 9 12 15
Control
PDJ- inoc+
PDJ+ inoc+
Control
PDJ+ inoc+
LSD0.05 = 14.4
PDJ− inoc+
Days after inoculation
JA(
nmol
/kg )
Chapter 2 Jasmonate application influences endogenous abscisic acid, jasmonic acid and aroma
volatiles in grapes infected by a pathogen (Glomerella cingulata)
41
Figure. 2.5. Changes in O2− radical scavenging activity in ‘Kyoho’ grapes after
pathogen inoculation and PDJ application. Data are the means ± SE of three
replications.
2.3.4 Aroma volatiles
Sixty-four kinds of aroma volatiles were detected in grape skins, including
17 aldehydes, 24 alcohols, 19 esters, 2 terpenes, and other small amount of
compounds (Table 2.5). At harvest, the aroma volatiles were comprised of an
abundance of aldehydes (90.9%), followed by alcohols (7.2%), esters (1.1%), and
terpenes (0.5%). (E)-2-hexenal (76.47 mg kg-1), hexanal (25.06 mg kg-1), and
0
1
2
3
4
5
6
7
0 3 6 9 12 15
Control PDJ- inoc+ PDJ+ inoc+
EC50(
mg
/mL )
Days after inoculation
LSD0.05 = 0.97Control
PDJ− inoc+
PDJ+ inoc+
Chapter 2 Jasmonate application influences endogenous abscisic acid, jasmonic acid and aroma
volatiles in grapes infected by a pathogen (Glomerella cingulata)
42
cis-3-hexenal (5.16 mg kg-1) were the primary compounds of aldehydes in ‘Kyoho’
grape skins. In general, aldehyde concentrations in PDJ− inoc+ were lower than in
PDJ+ inoc+ and the untreated control (Fig. 2.6). Alcohol concentrations increased
gradually with days after treatment, but were not significantly different between
treatments. The ester concentrations in PDJ+ inoc+ were higher than those in the
untreated control at 6 days after inoculation. Moreover, the concentrations of terpene
in PDJ+ inoc+ significantly increased in the 6 days after inoculation. The total aroma
volatiles reached a maximum at 9 days after inoculation.
Chapter 2 Jasmonate application influences endogenous abscisic acid, jasmonic acid and aroma
volatiles in grapes infected by a pathogen (Glomerella cingulata)
43
1
Table 2. Typical aroma volatile compounds identified in ‘Kyoho’ grape skins after pathogen inoculation and PDJ application.
Concentrations (mg kg-1)
Control PDJ
− inoc
+ PDJ
+ inoc
+
KIb Compounds 0 3 6 9 12 3 6 9 12 3 6 9 12
Alcohol
836 ethanol 2.1±0.5 13.9±4.6 16.6±0.0 34.9±1.8 38.2±6.9 5.8 ±1.0 9.5 ±1.6 41.6±4.4 47.0±10.0 6.8±2.2 14.8±1.4 33.4±3.4 47.5±4.8
1055 1-penten-3-
ol trc 0.6±0.3 1.5±0.2 2.2±0.9 1.0±0.2 0.6 ±0.2 1.1 ±0.1 1.1±0.2 0.8±0.1 1.7±0.2 2.3±0.2 1.5±0.2 1.0±0.2
1245 1-hexanol 0.3±0.0 0.6±0.1 0.5±0.1 0.7±0.1 0.9±0.1 0.9±0.2 0.4±0.0 0.6±0.1 0.6±0.1 0.8±0.0 1.0±0.2 0.7±0.1 0.6±0.1
1380 2-ethyl
-hexanol 2.3±0.2 2.0±0.4 2.2±0.4 2.6±0.3 1.1±0.5 2.7±0.6 3.0±0.7 3.1±0.5 2.2±0.2 2.0±0.1 3.2±0.4 4.8±1.9 3.8±0.8
Aldehyde
975 hexanal 25.1±4.8 47.7±4.6 46.1±6.9 60.0±12.1 38.1±6.5 22.7±4.9 34.5±3.9 40.7±6.6 37.9±1.2 36.3±4.3 51.1±2.5 65.3±5.2 43.6±5.8
1035 cis-3-
hexenal 5.2±2.3 6.9±1.4 4.6±0.3 2.6±0.4 1.5±0.4 2.8±0.2 2.5±0.9 3.0±0.3 0.3±0.1 3.5±0.6 3.4±0.2 4.9±1.5 1.3±0.0
1079 n-heptanal NDd 0.4±0.0 0.7±0.2 0.8±0.3 1.6±0.7 0.5±0.2 0.8±0.3 0.6±0.1 tr 0.7±0.0 1.0±0.3 1.5±0.8 0.9±0.2
1094 (Z)-2-
hexenal 1.3±0.2 2.2±0.8 1.5±0.2 1.2±0.2 0.8±0.1 0.6±0.2 0.9±0.0 1.1±0.2 0.6±0.2 1.0±0.2 1.1±0.2 1.3±0.1 0.7±0.2
1110 (E)-2-
hexenal 76.5±13.3 48.5±15.1 89.0±15.5 116.8±15.7 84.1±9.5 36.9±7.2 74.7±7.1 95.3±19.3 88.7±0.8 56.9±6.5 74.4±7.2 124.8±10.7 86.5±12.9
1289 nonanal 0.6±0.0 1.1±0.1 0.7±0.1 1.2±0.2 1.1±0.1 0.5±0.1 0.8±0.1 0.7±0.1 1.1±0.3 0.6±0.1 0.9±0.1 1.1±0.2 0.8±0.2
Ester
1020 hexyl
butanoate ND ND 0.4±0.1 0.6±0.2 0.5±0.1 0.8±0.1 0.6±0.1 0.3±0.1 tr 0.5±0.1 1.1±0.4 0.9±0.2 0.3±0.1
1030 ethyl
hexanoate 0.5±0.1 2.0±0.6 1.0±0.2 0.7±0.1 0.5±0.0 0.3±0.0 0.8±0.0 0.8±0.2 0.4±0.2 0.5±0.1 0.5±0.0 0.8±0.1 0.8±0.3
1305 3-heptene-1-
yl acetate tr 0.3±0.1 0.2±0.1 0.9±0.3 tr 2.1±0.9 0.5±0.2 0.6±0.4 2.9±0.4 5.3±2.8 4.1±2.4 2.6±0.9 0.2±0.1
1738
ethyl (E,Z)-
2,4-
decadienoate
ND 2.1±0.3 1.3±0.3 1.2±0.2 0.6±0.0 0.9±0.3 1.4±0.1 0.8±0.3 0.7±0.3 1.2±0.2 1.1±0.1 1.3±0.3 0.9±0.3
Others
1449 α-Farnesene 0.2±0.0 0.5±0.1 0.3±0.0 0.6±0.1 0.5±0.1 0.3±0.1 0.3±0.0 0.4±0.1 0.4±0.0 0.3±0.0 0.4±0.1 0.5±0.1 0.4±0.0
1231
6-methyl-5-
hepten-2-
one
0.3±0.1 0.5±0.1 0.5±0.1 0.5±0.0 0.6±0.1 1.1±0.2 0.3±0.0 0.4±0.1 0.5±0.0 1.2±0.2 1.4±0.4 0.6±0.1 0.5±0.1
a Data are the means ± SE of three replications. b n-Alkanes were analysed under the same conditions to calculate Kovats Index (KI) for the volatile compounds. c tr = relative concentration less than 0.2
mg kg-1
. d ND = relative concentration less than 0.1 mg kg-1
a
Chapter 2 Jasmonate application influences endogenous abscisic acid, jasmonic acid and aroma
volatiles in grapes infected by a pathogen (Glomerella cingulata)
44
Fig. 2.6. The concentration of volatile compounds [(A) Aldehydes concentrations; (B)
Alcohol concentrations; (C) Ester concentrations; (D) Terpene concentrations] in
‘Kyoho’ grape skins after pathogen inoculation and PDJ application. Data are the
means ± SE of three replications.
0
50
100
150
200
250 Control PDJ-inoc- PDJ+inoc-
Ald
ehyd
e co
ncen
tratio
n��(m
g/kg
) �
LSD0.05= 35.70�
(A)�
PDJ− inoc+ PDJ+ inoc+ Control
0
10
20
30
40
50
60
70
80
Alc
ohol
con
cent
ratio
n��(m
g/kg
)�
LSD0.05= 9.66�
(B)�
0
2
4
6
8
10
12
14
16
0 3 6 9 12 15
Este
r con
cent
ratio
n��(m
g/kg
)�
Days after inoculation�
LSD0.05= 3.36� (C)�
0
1
2
3
4
0 3 6 9 12 15
��
T
erpe
ne c
once
ntra
tion��(m
g/kg
) �
Days after inoculation�
LSD0.05= 0.52�(D)�
Chapter 2 Jasmonate application influences endogenous abscisic acid, jasmonic acid and aroma
volatiles in grapes infected by a pathogen (Glomerella cingulata)
45
2.4 DISCUSSION
Some reports have shown a positive correlation between ABA levels and
disease resistance. Viral infection increased ABA concentrations, and ABA
application increased virus resistance in tobacco (Nicotiana tabacum L.) (Fraser et
al., 1982; Whenham et al., 1986). ABA treatment enhanced basal resistance of rice
(Oryza sativa L.) against the brown spot-causing ascomycete Cochliobolus
miyabeanus (Vleesschauwer et al. 2010). In addition, cds2-1D mutants,
which overexpress 9-cis epoxycarotenoid dioxigenase5 (NCED5; one of the six
genes encoding the ABA biosynthetic enzyme NCED), show enhanced endogenous
ABA and increased resistance against Alternaria brassicicola. In contrast,
ABA-deficient mutant aba3-1 did not have resistance against A. brassicicola in
Arabidopsis (Arabidopsis thaliana) (Fan et al., 2009). In my study, the increased
concentrations of ABA in inoculated fruit suggest that ABA might be associated with
the defense system in grape berries. Environmental stress regulates ABA
biosynthesis in plants. For instance, the expression of NCED in Arabidopsis (Iuchi et
al. 2001), cowpea (Vigna unguiculata L.) (Iuchi et al., 2000), and avocado (Persea
americana L.) (Chernys et al., 2000) were stimulated by drought stress. In this study,
inoculation also induced the expression of VvNCED1. Therefore, it is possible that
the increase in ABA concentrations in PDJ+ inoc+ were dependent on the increased
Chapter 2 Jasmonate application influences endogenous abscisic acid, jasmonic acid and aroma
volatiles in grapes infected by a pathogen (Glomerella cingulata)
46
expression of VvNCED1 gene. Moreover, it is thought that the increase of PA was
induced by the VvCYP707A gene encoding the ABA 8′-hydroxylase. In addition,
PDJ treatment increased the ABA concentrations in apples (Malus sylvestris L.)
(Kondo et al., 2000). These results suggest cross-talk between jasmonate and ABA,
including VvNCED1 and VvCYP707A1.
Synergistic and antagonistic effects have been reported in the interaction
between the ABA and JA signaling pathways (Moons et al., 1997; Staswick et al.,
1992). For example, high ABA levels strongly reduced the transcript levels of JA- or
ethylene-responsive defense genes in Arabidopsis inoculated by Fusarium
oxysporum, whereas those in ABA-deficient mutants increased (Anderson et al.
2004). On the other hand, ABA stimulated biosynthesis of JA and the JA-dependent
defense gene in aba2-12 biosynthetic defective mutants in Arabidopsis against
Pythium irregular (Adie et al., 2007). In my study, PDJ application stimulated
endogenous ABA and JA concentrations, resulting in a decrease of lesion diameter
by pathogen infection. Therefore, the defense mechanism observed in grape berries
may depend on the synergistic effect of JA and ABA.
In this study, aldehydes and alcohols were primarily detected in abundance
during storage among all treatments, as they were in previous research (Hadi et al.,
2013), showing that volatile compounds in grape berries predominantly consist of
short chain aldehydes and alcohols. (E)-2-hexenal was the most abundant volatile
Chapter 2 Jasmonate application influences endogenous abscisic acid, jasmonic acid and aroma
volatiles in grapes infected by a pathogen (Glomerella cingulata)
47
compound in ‘Riesling’ and ‘Cabernet Sauvignon’ grapes, and showed a significant
increase after véraison (Kalua et al., 2010). It has been shown that trans-2-hexenal,
carvacrol, trans-cinnamaldehyde, and citral provided consistent fungicidal activity
against Penicillium expansum in pears (cv. Conference) (Neri et al., 2006). In my
study, (E)-2-hexenal was the major volatile. The plant defense response on C6
aldehydes ((E)-2-hexenal and hexenal in our study) have been shown. For example,
they are the predominant wound-inducible volatiles that mediate indirect defense
responses by attracting the natural enemies against plant invaders (Kessler et al.,
2004; Farag et al., 2005). In showing that aldehyde concentrations in PDJ+ inoc+ were
higher than in PDJ− inoc+, this study corroborates these reports. Previous research
showed that PDJ application at the pre-climacteric stage increased the production of
esters in apples (Kondo and Matheis, 2006). Ester formation in grapes needs a supply
of substrate (alcohols) and the ester-forming enzyme, alcohol acyltransferase (AAT)
(Wang and Luca, 2005). As the ester concentration significantly increased in PDJ+
inoc+, PDJ application might have influenced the accumulation of esters by the
stimulating activity of AAT. The terpenes or terpenoids constitute the largest class of
secondary products that appear to play important defensive roles in plants
(Gershenzon et al., 1992). A significant increase of terpenes 6 days after inoculation
was detected in PDJ+ inoc+.
Ethanol has been considered germistatic against fruit and vegetable decay
Chapter 2 Jasmonate application influences endogenous abscisic acid, jasmonic acid and aroma
volatiles in grapes infected by a pathogen (Glomerella cingulata)
48
microorganisms (Penicillium digitatum and Colletotrichum musae in an in vitro
experiment) (Utama et al., 2002). In my study, ethanol was the most-identified
compound in the alcohol class, and it was derived from fermentation, which
transforms to ethanol through the action of the enzyme alcohol dehydrogenase
(ADH) (William et al. 2008). ADH enzyme activity and HvADH1 were stimulated
by the inoculation of Blumeria graminis in barley (Hordeum vulgare L.) (Pathuri et
al., 2011). Therefore, ethanol production may also be a signal that plants are
resistant to pathogen infection. In my study, although alcohol concentrations
increased in PDJ+ inoc+ compared to PDJ− inoc+, there were no significant
differences with the untreated control. The aspect of aroma volatile emission in
plant defense mechanism may be different among plants.
In summary, endogenous ABA and JA concentrations increased in
inoculated ‘Kyoho’ grape berries. PDJ application before inoculation increased
ABA concentration, as well as the expression of the VvNCED1 gene. The defense
mechanism in grape berries against pathogenic infection might depend on the
synergistic effect of JA and ABA.
Chapter 3 α-Ketol linolenic acid (KODA) application affects endogenous abscisic acid, jasmonic acid
and aroma volatiles in grapes infected by a pathogen (Glomerella cingulata)
49
CHAPTER 3
α-KETOL LINOLENIC ACID (KODA) APPLICATION
AFFECTS ENDOGENOUS ABSCISIC ACID, JASMONIC
ACID, AND AROMA VOLATILES IN GRAPE BERRIES
INFECTED BY A PATHOGEN
(GLOMERELLA CINGULATA)
Chapter 3 α-Ketol linolenic acid (KODA) application affects endogenous abscisic acid, jasmonic acid
and aroma volatiles in grapes infected by a pathogen (Glomerella cingulata)
50
3.1 INTRODUCTION
G. cingulata is a plant pathogenic fungus that causes diseases as bitter rot
and anthracnose in many fruit and vegetable species. It has been shown that plant
hormones stimulate physiological response particularly under environmental stress
such as drought, salt and disease (Yamaguchi-Shinozaki and Shinozaki, 1993;
Albacete et al., 2008; Denancé et al., 2013). KODA: [9,10-ketol-octadecadienoic
acid or 9-hydroxy-10-oxo-12(Z), 15(Z)-octadecadienoic acid] was identified as a
stress-induced substance in duckweed (Lemna paucicostata) (Yokoyama et al., 2000).
KODA reacted as a flower-inducing compound in violet (Pharbitis nil), carnation
(Dianthus caryophyllus L.), and apple (Malus domestica Borkh.) (Kittikorn et al.,
2010; Suzuki et al., 2003; Yokoyama et al., 2005).
KODA is formed from 9-hydroperoxy-10(E), 12(Z),
15(Z)-octadecatrienoic acid (9-HPOT) generated from linolenic acid via
9-lipoxygenase (9-LOX) (Vick and Zimmerman, 1987). JA, which its regulatory
function in defensing fungal pathogen was well demonstrated, is synthesized from
linolenic acid by 13-hydroperoxy-9(Z), 11(E), 15(Z)-octadecatrienoic acid
(13-HPOT) by 13-LOX (Turner et al., 2002; Vick and Zimmerman 1987). In
oxylipin pathway, both KODA and JA are metabolized via LOX and AOS, known as
AOS pathway (Creelman and Mullet, 1997; Yamaguchi et al., 2001). The other is the
Chapter 3 α-Ketol linolenic acid (KODA) application affects endogenous abscisic acid, jasmonic acid
and aroma volatiles in grapes infected by a pathogen (Glomerella cingulata)
51
fatty acid HPL pathway that produces C6-aldehydes and C12-oxo acids (Hatanaka et
al., 1987). Volatile C6-aldehydes, (E)-2-hexenal and (Z)-3-hexenal enhance
resistance against a necrotrophic pathogen (Botrytis cinerea) in Arabidopsis
(Kishimoto et al., 2006). In view of these achievements, KODA might influence the
infection of pathogen was also involved in our study, which is absolutely unclear.
ABA, as a regulator in ripening grape berries, has been shown to act
complex role to response against environmental stress such as drought via stomatal
closure and pathogen attack by interacting with other plant hormones (Coombe and
Hale, 1973; Mohr and Cahill, 2001; Yamaguchi-Shinozaki and Shinozaki, 1993). In
the ABA biosynthetic pathway of higher plant, the NCED is a key upstream enzyme
that cleaves 11, 12 double bonds of C40 carotenoids and produces xanthoxin (Qin and
Zeevaart, 1999). Then, ABA primarily catabolizes to 8′ -hydroxy ABA by ABA
8′-hydroxylase, and subsequently isomerizes to PA (Nambara and Marion-Poll,
2005). SA was also key signal molecules in the activation of plant defense responses
as well as JA (Delaney et al., 1994). SA had a negative effect on trichome
production and consistently reduced the effect of JA in Arabidopsis, suggesting
negative cross-talk between the JA and SA-dependent defense pathways (Traw and
Bergelson, 2003). Nevertheless, any clues in interaction between JA and SA have not
been acquired in grape berries after infection of G. cingulata.
It is possible that plant defense in response to microbial attack is regulated
Chapter 3 α-Ketol linolenic acid (KODA) application affects endogenous abscisic acid, jasmonic acid
and aroma volatiles in grapes infected by a pathogen (Glomerella cingulata)
52
through a complex network of signaling pathways that might involve ABA, JA, SA,
and C6-aldehydes, which would be discussed in our research.
Chapter 3 α-Ketol linolenic acid (KODA) application affects endogenous abscisic acid, jasmonic acid
and aroma volatiles in grapes infected by a pathogen (Glomerella cingulata)
53
3.2 MATERIALS AND METHODS
3.2.1. Plant material
Three 8-year-old ‘Kyoho’ [Vitis labrusca × V. vinifera] grapevines grafted
onto ‘Teleki-kober 5BB’ rootstocks [V. berlandieri × V.riparia hibrids] were
harvested at the véraison period (50 days after full bloom [DAFB]). The vines had
been cultivated in an open field at Chiba University, located at 35 °N Lat., 140 °E
Long., and at an altitude of 37 m. Two times of 25ppm GA treatment were applied
for the development and seedless of grape berries (First time: at full blood date;
Second time: 10 days after full bloom). Berries were selected uniformly in size and
color and rinsed with tap water, then sterilized by 0.5% sodium hypochlorite for 3
min and washed with distilled water prior to KODA treatment. The fruits were
randomly divided into three groups, 800 berries of each. The first and second groups
of fruits were immersed in the distilled water containing 0.1% (v/v) surfactant
Approach BI (50% polyoxyethylene hexitan fatty acid ester; Kao, Osaka, Japan) for
5 min. The third group, the fruits were dipped into 0.1 mM KODA solution (Shiseido,
Co., Ltd, Tokyo, Japan), containing 0.1% (v/v) Approach BI for 5 min. After air-dry,
all of the berries were wounded with the needle of a syringe to a depth of 5 mm.
Fruits in the second and third groups were inoculated with G. cingulata.
Chapter 3 α-Ketol linolenic acid (KODA) application affects endogenous abscisic acid, jasmonic acid
and aroma volatiles in grapes infected by a pathogen (Glomerella cingulata)
54
3.2.2. Fungal strain, culture, and inoculation.
The G. cingulate (MAFF, 239927) applied in our research was incubated
on potato dextrose agar at 25°C for 7 days. The conidia were collected with distilled
water after the surface of the medium was scraped. Density of the conidia suspension
was adjusted to 106 conidia/mL with distilled water using a Thoma’s hemacytometer
(EKDS Co., Tokyo, Japan).
A drop of suspension with spores was dripped on the wounded surfaces of
each fruit in the second and third groups, hereafter referred to as KODA− inoc+ and
KODA+ inoc+, respectively. The control fruits were treated with suspension without
spores by the same way, hereafter referred to as the control.
All the berries after treatment were stored in a controlled room at 25°C
with 95% relative humidity. Two hundred berries from each treatment (three
replications of 66-67 berries) were sampled at 3, 6, 9, and 12 days after inoculation.
Two hundred berries (three replications of 66-67 berries) were collected immediately
after harvest prior to treatment (day 0). After lesion diameter and ethylene
production were analyzed, the peel was sampled and frozen with liquid N2, then
stored at −80°C immediately.
3.2.3 Quantitative analysis of ABA and PA
The measurement of ABA and PA followed the method described in 2.2.3.
Chapter 3 α-Ketol linolenic acid (KODA) application affects endogenous abscisic acid, jasmonic acid
and aroma volatiles in grapes infected by a pathogen (Glomerella cingulata)
55
3.2.4 Jasmonic acid analysis
The analysis of JA was done by GC-MS-SIM described in 2.2.4.
3.2.5. Volatile compound analysis
Aroma volatile compound in frozen skin samples were extracted and
measured followed 2.2.5.
3.2.6 Measurement of O2− radical-scavenging activity
The analysis of O2− radical-scavenging activity were measured as previous
method described by Kondo et al. (2005). One gram of sample (three replications)
were homogenized with 10 mL 13.7 mol/L ethanol, filtered and evaporated to the
aqueous phase, and added to 10 mL of distilled water.
3.2.7 RNA extraction, cDNA synthesis, and quantitative real-time RT-PCR
analysis
Total RNA was extracted by a cetyltrimethylammonium bromide
(CTAB)-based method from skin sample (1 g FW; three replications) previously
reported by Kondo et al. (2012). cDNA synthesis followed the instruction manual of
ReverTra Ace® qPCR RT Master Mix (Code No. FSQ-201) (Toyobo co., LTD.,
Osaka, Japan). Quantitative PCR was performed on StepOnePlus™ system (Applied
Chapter 3 α-Ketol linolenic acid (KODA) application affects endogenous abscisic acid, jasmonic acid
and aroma volatiles in grapes infected by a pathogen (Glomerella cingulata)
56
Biosystems, SMFC, CA, USA). The primers listed in Table 3.1 were used for Q-PCR.
Primers of the VvCYP707A1 gene were according to a previous report (Kondo et al.
2014). Primers for the VvNCED1 gene were constructed based on the sequences
reported by Sun et al. (2010). Besides, VvLOX and VvAOS involved in oxylipin
pathway were designed as described by Ramírez-Suero et al. (2014). The relative
expression level was determined with the VvUbiquitin as an internal control gene by
a relative standard curve method (Fujita et al. 2007).
Tables 3.1 Primers used for Quantitative real-time RT-PCR.
Gene Forward/reverse primer (5′ – 3′ ) Reference
VvNCED1 (F) GGTGGTGAGCCTCTGTTCCT Sun et al. (2010)
(R) CTGTAAATTCGTGGCGTTCACT
VvCYP707A1 (F) GAAACATTCACCACAGTCCAGA Kondo et al.
(2014) (R) AGCAAAAGGGCCATACTGAATA
VvAOS (F) GCCTGGCTTAATCACGACAT
Ramírez-Suero et
al. (2014) (R) CACCTTCGTCCAGAACATGA
VvLOX (F) CCCTTCTTGGCATCTCCCTTA
Ramírez-Suero et
al. (2014) (R) TGTTGTGTCCAGGGTCCATTC
VvUbiquitin (F) TCTGAGGCTTCGTGGTGGTA Fujita et al.
(2007) (R) AGGCGTGCATAACATTTGCG
Chapter 3 α-Ketol linolenic acid (KODA) application affects endogenous abscisic acid, jasmonic acid
and aroma volatiles in grapes infected by a pathogen (Glomerella cingulata)
57
3.2.8 Statistical analysis
The each data was presented as the mean values of the three replications ±
the standard error (SE), subjected to analysis of variance procedures, and separated
by Turkey-Kramer test at P ≤ 0.05 using the SAS statistical analysis package
(version 8.2, SAS Institute, Cary, NC, USA). The lesion diameter in Fig. 3.1 was
evaluated using t-test on each day after treatment (**, P ≤ 0.01).
Chapter 3 α-Ketol linolenic acid (KODA) application affects endogenous abscisic acid, jasmonic acid
and aroma volatiles in grapes infected by a pathogen (Glomerella cingulata)
58
3.3 RESULTS
3.3.1. Lesion diameter
The diameters of the lesions in grape samples infected by G. cingulata
were exhibited in Fig. 3.1. Lesion diameter in KODA+ inoc+ samples were
significantly suppressed than those in KODA− inoc+ from 6 days, although reverse
result was presented at 3 days after inoculation.
Fig. 3.1. Lesion diameter during storage in ‘Kyoho’ grape berries of pathogen
inoculation without KODA application and pathogen inoculation with KODA
application. KODA solution was applied 24 h prior to inoculation. Data are the
means from three replications of 50 berries. Asterisks indicate significant differences
by using t-test on each day after treatment (**, P ≤ 0.01).
0
5
10
15
20
25
0 3 6 9 12
KODA+inoc+ KODA+inoc+
Lesi
on d
iam
eter
(mm
)
Days after inoculation
**
**
****
KODA+ inoc+ KODA− inoc+
Chapter 3 α-Ketol linolenic acid (KODA) application affects endogenous abscisic acid, jasmonic acid
and aroma volatiles in grapes infected by a pathogen (Glomerella cingulata)
59
3.3.2. Endogenous abscisic acid (ABA), phaseic acid (PA) and expression of
VvNCED1 and VvCYP707A1
ABA levels in inoculated fruit increased significantly at 3 days and
reduced since 6 days after infection (Fig. 3.2). Moreover, the ABA concentrations
in KODA+ inoc+ were higher than those in KODA− inoc+ at 6 and 9 days after
inoculation. PA concentrations also increased after inoculation, especially were
highest in PDJ+ inoc+ at 6 and 9 days after inoculation. The expression levels of
VvNCED1 in KODA+ inoc+ were highest at 6 days after inoculation, compared to
KODA− inoc+ and the untreated control, which is accordance with the variation of
ABA concentration. The expressions of VvCYP707A1 in KODA+ inoc+ were highest
at 6 days after inoculation, which is consistent with PA levels.
3.3.3 Endogenous jasmonic acid (JA), expression of VvLOX and VvAOS,
ethylene production, and the changes in the O2− radical scavenging activities
JA concentrations in KODA+ inoc+ were higher than those in untreated
control and KODA− inoc+ at 9 days after inoculation (Fig. 3.3). The expression of
VvLOX and VvAOS were up-regulated significantly in KODA+ inoc+ than those in
KODA− inoc+ and untreated control samples at 6, 9, and 12 days after inoculation.
Ethylene production did not show significant differences among all the treatments
(data not shown). The O2− scavenging activities, presented as half maximal (50%)
Chapter 3 α-Ketol linolenic acid (KODA) application affects endogenous abscisic acid, jasmonic acid
and aroma volatiles in grapes infected by a pathogen (Glomerella cingulata)
60
effective concentration (EC50), which was defined as the concentration of substrate
that caused a 50% loss in O2− scavenging activities. Low EC50 values exhibit high
antioxidant activity. The EC50 values in KODA+ inoc+ were lowest at 6 and 9 days
after inoculation (Fig. 3.4).
Chapter 3 α-Ketol linolenic acid (KODA) application affects endogenous abscisic acid, jasmonic acid
and aroma volatiles in grapes infected by a pathogen (Glomerella cingulata)
61
Fig. 3.2. Endogenous (A) ABA and (B) PA concentrations, and quantitative real
time RT-PCR analysis of (C) VvNCED1 and (D) VvCYP707A1 in ‘Kyoho’ grape
skins after pathogen inoculation and KODA application. KODA solution was applied
24 h prior to inoculation. Data are the means ± SE of three replications. Different
letters indicate significant differences by Tukey-Kramer test at P ≤ 0.05. ns, not
significant.
Chapter 3 α-Ketol linolenic acid (KODA) application affects endogenous abscisic acid, jasmonic acid
and aroma volatiles in grapes infected by a pathogen (Glomerella cingulata)
62
Fig. 3.3 Quantitative real time RT-PCR analysis of (A) VvLOX and (B) VvAOS, and
endogenous (C) JA concentrations in ‘Kyoho’ grape skins after pathogen inoculation
and KODA application. KODA solution was applied 24 h prior to inoculation. Data
are the means ± SE of three replications. Different letters indicate significant
differences by Tukey-Kramer test at P ≤ 0.05.
0
50
100
150
200
250
300
350
0 3 6 9 12
JA (n
mol
/kg)
Days after inoculation (d)�
ns�a�
ns�
a�
b�ab�
b�
b�
(C)�
Chapter 3 α-Ketol linolenic acid (KODA) application affects endogenous abscisic acid, jasmonic acid
and aroma volatiles in grapes infected by a pathogen (Glomerella cingulata)
63
Fig. 3.4. Changes in O2− radical scavenging activity in ‘Kyoho’ grape skins of
untreated control (n), pathogen inoculation without KODA application (●), and
pathogen inoculation with KODA application (▲). KODA solution was applied 24 h
prior to inoculation. Data are the means ± SE of three replications. Different letters
indicate significant differences by Tukey-Kramer test at P ≤ 0.05.
Chapter 3 α-Ketol linolenic acid (KODA) application affects endogenous abscisic acid, jasmonic acid
and aroma volatiles in grapes infected by a pathogen (Glomerella cingulata)
64
3.3.4 Aroma volatiles
There were 67 kinds of aroma volatiles detected in grape skins, including
17 aldehydes, 26 alcohols, 18 esters, 2 terpenes and other trace of compounds (Table
3.2). Before inoculation (day 0), the totals of aroma volatiles were 152.6 mg kg-1, in
which quantities of aldehydes (78.1%) were highest, followed by alcohols (8.9%),
esters (11.1%), terpenes and ketone (1.0%). Compounds of aldehydes were primarily
comprised of C6-aldehydes including (E)-2-hexenal (73.4 mg kg-1), hexanal (36.7
mg kg-1), and cis-3-hexenal (6.8 mg kg-1) in ‘Kyoho’ grapes. The total of aroma
volatiles were 165.5 mg kg-1 in KODA+ inoc+,164.8 mg kg-1 in KODA− inoc+ and
194.1 mg kg-1 in the untreated control at 12 days after inoculation, respectively. The
total aroma volatiles reached a maximum in KODA+ inoc+ (260.4 mg kg-1) 6 days
after inoculation. Aldehyde concentrations in KODA+ inoc+ were significantly higher
than those in KODA− inoc+ and the untreated control at 6 and 9 days after inoculation
(Fig. 3.5A). The alcohol and ester concentrations in KODA+ inoc+ were significantly
higher than those in KODA− inoc+ and the untreated control at 3 and 6 days after
inoculation (Fig. 3.5B and C). Methyl salicylate (Me-SA) concentrations in KODA+
inoc+ were higher than those in other treatments at 6 and 9 days after inoculation (Fig.
3.5C). In addition, the concentrations of terpenes (α-Farnesene and its oxidation
product 6-methyl-5-hepten-2-one) in KODA+ inoc+ were highest after inoculation
(Fig. 3.5D).
Chapter 3 α-Ketol linolenic acid (KODA) application affects endogenous abscisic acid, jasmonic acid
and aroma volatiles in grapes infected by a pathogen (Glomerella cingulata)
65
Table 3.2. Primal aroma volatile compounds in ‘Kyoho’ grape skins after pathogen inoculation and KODA applicationa.
Concentrations (mg kg-1)
Control KODA−
inoc+
KODA+
inoc+
KIb Compounds 0 3 6 9 12 3 6 9 12 3 6 9 12
Alcohol
835 ethanol 3.0±0.4 12.9±0.5 10.9±1.9 15.2±0.3 26.3±4.6 5.8 ±1.0 9.5 ±1.6 33.4±3.
4 47.0±10.
0 6.8±2.2 14.8±1.4 41.6±4.4 47.5±4.8
1055 1-penten-3-ol trc 0.4±0.0 0.2±0.1 0.3±0.0 0.7±0.2 tr 0.2±0.0 1.4±0.6 0.8±0.1 0.4±0.1 NDd 0.4±0.2 1.6±0.0
1099
2-methyl-1-
butanol
1.0±0.3 0.6±0.2 0.9±0.0 1.1±0.1 1.1±0.1 0.9±0.1 1.1±0.2 1.0±0.3 1.3±0.2 0.8±0.1 1.4±0.1 0.8±0.1 1.5±0.1
1211 (Z)-2-penten-1-ol 0.7±0.1 0.7±0.0 0.4±0.0 0.9±0.0 1.0±0.3 0.7±0.1 0.7±0.2 1.3±0.2 0.7±0.2 0.5±0.1 1.0±0.1 0.8±0.2 0.9±0.3
1258 1-hexanol 0.8±0.1 1.2±0.0 0.6±0.1 0.8±0.1 0.6±0.0 1.2±0.2 1.1±0.2 0.6±0.1 0.9±0.1 0.8±0.0 2.1±0.2 1.6±0.1 0.9±0.1
1278 (Z)-3-hexen-1-
ol
0.8±0.1 0.6±0.0 0.5±0.1 ND 0.3±0.1 0.7±0.1 0.7±0.1 ND 0.4±0.2 0.9±0.0 1.0±0.1 0.9±0.2 0.2±0.1
1380 2-ethylhexanol 2.5±0.5 3.8±0.1 3.0±0.4 7.0±0.4 5.6±0.4 4.8±0.8 3.7±0.6 6.2±1.1 6.1±1.0 6.3±1.1 9.3±1.5 6.3±1.2 8.2±2.0
1801 Phenylethyl alcohol 0.4±0.1 0.6±0.0 0.5±0.0 1.6±0.2 1.9±0.2 0.6±0.1 0.7±0.1 1.5±0.2 2.0±0.1 0.7±0.1 1.9±0.1 0.7±0.2 2.7±0.4
Aldehyde
975 hexanal 36.7±6.7 44.6±0.4 40.7±4.0 40.0±2.4 37.0±1.8 39.9±8.3 39.0±6.0 29.4±2.
0 28.3±4.0 51.8±0.7 66.3±2.1 51.8±4.2 39.9±6.0
1035 cis-3-hexenal 6.8±1.8 6.2±0.6 3.1±0.3 1.2±0.4 1.8±0.2 2.8±0.2 2.8±0.5 2.0±0.1 0.6±0.3 2.9±0.8 5.0±0.2 2.9±0.2 2.6±0.2
1093 (Z)-2-hexenal ND 1.4±0.1 0.8±0.0 0.3±0.0 0.7±0.1 1.0±0.2 1.2±0.2 ND 0.3±0.1 1.1±0.1 1.5±0.1 1.1±0.0 0.6±0.1
1109 (E)-2-hexenal 73.4±11.7 66.7±2.1 68.0±9.1 62.1±5.9 55.6±1.
2 69.4±11.2
70.6±11.2
50.7±5.7 38.2±3.6 81.1±1.9 124.4±8.
5 81.1.8±4
.3 57.1±7.3
1288 nonanal 0.7±0.1 0.6±0.0 0.9±0.1 0.6±0.1 0.6±0.1 0.5±0.0 0.6±0.1 0.6±0.0 0.4±0.0 0.8±0.1 1.0±0.1 0.8±0.0 0.8±0.2
1539 Benzeneacetal
-dehyde
0.4±0.1 0.3±0.0 0.4±0.1 0.8±0.1 0.7±0.1 0.4±0.1 0.4±0.1 0.6±0.1 0.7±0.0 0.3±0.1 0.9±0.1 0.3±0.1 1.0±0.1
Ester
1019 hexyl butanoate 0.5±0.1 0.2±0.2 0.8±0.1 1.2±0.4 0.7±0.1 0.8±0.2 0.8±0.1 0.7±0.3 1.0±0.4 0.7±0.2 0.4±0.3 1.0±0.2 1.7±0.1
1228 (E)-2-hexenyl
acetate
tr tr ND 1.4±0.4 1.8±0.3 tr ND 0.7±0.6 2.5±0.6 0.7±0.3 0.3±0.0 1.3±0.2 1.8±0.4
1242 ethyl 2-hexenoate
ND ND ND 1.7±0.2 1.7±0.1 ND ND tr 2.2±0.2 tr tr 1.5±0.1 2.2±0.4
1304 3-heptene-1-yl acetate 2.1±0.4 1.1±0.3 tr 5.9±3.2 13.5±4.
7 1.4±0.9 0.3±0.1 7.8±4.8 20.9±7.5 7.8±5.2 2.2±2.4 5.9±2.1 10.7±4.0
1683 methyl salicylate
ND 0.6±0.0 0.3±0.1 0.3±0.1 0.3±0.0 1.1±0.1 0.4±0.2 0.5±0.1 0.2±0.0 0.5±0.1 1.2±0.3 1.0±0.1 0.4±0.1
1743 ethyl
(E,Z)-2,4-decadienoate
tr 1.0±0.0 2.0±0.5 0.8±0.2 0.7±0.1 1.3±0.2 0.8±0.2 2.0±0.2 0.6±0.1 2.0±0.6 1.7±0.1 0.9±0.2 0.7±0.3
Terpene
1456 α-Farnesene 0.4±0.1 0.6±0.1 0.4±0.0 0.6±0.1 0.7±0.0 0.7±0.1 0.6±0.1 0.5±0.1 0.5±0.1 0.8±0.1 1.1±0.1 0.8±0.1 0.6±0.1
1234
6-methyl-5-hepten-2-one 0.3±0.1 0.3±0.0 0.3±0.1 0.6±0.2 0.7±0.0 0.3±0.1 0.2±0.0 0.6±0.1 1.0±0.2 0.6±0.1 0.5±0.1 0.6±0.2 0.9±0.2
a Data are the means ± SE of three replications. b n-Alkanes were analyzed under the same conditions to calculate Kovats Index (KI) for the volatile compounds. c tr = relative concentration less than
0.2 mg kg-1
. d ND = relative concentration less than 0.1 mg kg-1
.
Chapter 3 α-Ketol linolenic acid (KODA) application affects endogenous abscisic acid, jasmonic acid
and aroma volatiles in grapes infected by a pathogen (Glomerella cingulata)
66
Fig. 3.5. The concentration of volatile compounds [(A) Aldehydes concentrations; (B)
Alcohol concentrations; (C) Ester concentrations; (D) Terpene concentrations] in
‘Kyoho’ grapes of untreated control (n), pathogen inoculation without KODA
application (●) and pathogen inoculation with KODA application (▲). KODA
solution was applied 24 h prior to inoculation. Data are the means ± SE of three
replications. Different letters indicate significant differences by Tukey-Kramer test at
P ≤ 0.05.
Chapter 3 α-Ketol linolenic acid (KODA) application affects endogenous abscisic acid, jasmonic acid
and aroma volatiles in grapes infected by a pathogen (Glomerella cingulata)
67
3.4 DISCUSSION
It has been shown that mechanical injury to leaf tissue in potato plants
(Solanum tuberosum) caused an increase in ABA and JA which were also observed
in our study (Dammann et al., 1997). The JA or ABA initiates the accumulation of
wound-inducible gene proteinase inhibitor II (pin2) in tomato and potato plants,
suggesting that the presence of a complex network for regulating the effects of the
plant hormones ABA and JA on gene expression upon wounding (Pena-Cortes et al.,
1995).
KODA application did not retard the pathogen spreading on the agar
medium (data not shown), which suggests that KODA treatment may increase the
tolerant activity by inducing second metabolite against pathogen infection in grape
berries. Allene oxide synthase (AOS) is a cytochrome P-450 (CYP74A) that
catalyzes the first step in the conversion of 13-hydroperoxy linolenic acid to
jasmonic acid and 9-hydroperoxy linolenic acid to KODA (Itoh et al., 2002). In our
study, KODA application induced accumulation of JA by activating the VvLOX and
VvAOS expression. It has been shown that pathogen-induced modulation of signaling
via phytohormones contributes to virulence (Robert-Seilaniantz et al., 2011). For
example, ABA is not only important for mediating abiotic stress responses but also
plays a multifaceted and pivotal role in plant immunity (Cao et al., 2011).
Chapter 3 α-Ketol linolenic acid (KODA) application affects endogenous abscisic acid, jasmonic acid
and aroma volatiles in grapes infected by a pathogen (Glomerella cingulata)
68
Meta-analysis of transcriptome profiles confirmed the role of JA in activation of P.
irregulare–induced defenses and ABA as an important regulator of defense gene
expression, which indicated the synergetic interaction of ABA and JA (Adie et al.,
2007). It has been shown that ABA may suppress SA biosynthesis and SA-mediated
defense responses (Audenaert et al., 2002; Thaler and Bostock, 2004). Whereas,
ABA-mediated stress signals regulated SA biosynthesis by inducing the SA
biosynthetic enzyme gene SID2 in Arabidopsis (Seo and Park, 2010), which shows
ABA signaling might act synergistically with SA signaling in triggering plant
immune response. Me-SA, which is a volatile ester converted from SA, were higher
in KODA+ inoc+ than those in KODA− inoc+ and the untreated control. Rojo et al.
(2003) and Takahashi et al. (2004) showed the antagonistic interaction between SA
and JA signaling pathway. However, Schenk et al. (2000) and Mur et al. (2006)
revealed the synergetic action of SA and JA in response to pathogens, which is in
accordance with our result. Volatile C6-aldehydes such as (E)-2-hexenal, and
(Z)-3-hexenal are important in defense against microbial pathogen and insect attacks
(Engelberth et al., 2004; Kishimoto et al., 2006). The activities of the key enzymes,
LOX and HPL in production of C6-aldehydes were determined in many species such
as strawberry (Fragaria × ananassa Duch.) and soybean (Glycine max L.) (Myung
et al., 2006; Zhuang et al., 1992). In my study, VvLOX encoded LOX enzyme was
induced in KODA+ inoc+, which suggest that C6 -aldehydes were accumulated by
Chapter 3 α-Ketol linolenic acid (KODA) application affects endogenous abscisic acid, jasmonic acid
and aroma volatiles in grapes infected by a pathogen (Glomerella cingulata)
69
activation of VvLOX in grape. Chalcone synthase (CHS), caffeic
acid-O-methyltransferase (COMT), diacylglycerol kinase1 (DGK1),
glutathione-S-transferase1 (GST1) and lipoxygenase2 (LOX2), which are induced by
jasmonate application, are induced with C6 -aldehydes against Botrytis cinerea in
Arabidopsis. Therefore, the interaction of KODA, JA and C6-aldehydes were
involved in oxylipin pathway (Kishimoto et al., 2005). C6-aldehyses can be
transformed to the corresponding alcohols and esters through the activity of ADH
and alcohol acyltransferase (AAT), respectively (D’Auria et al., 2007; Hamberg et
al., 1999). It has been shown that ADHs are dependent medium-chain
dehydrogenases that are involved in the response to a wide range of stresses,
including anaerobiosis and pathogen (Chase, 2000; Proels et al., 2011).
α-Farnesene is emitted by a number of plant tissues in response to herbivory,
wounding, pathogen and general defense. For instance, tobacco plants challenged
with Pseudomonas syringae bacteria increase the production of α-farnesene and
MeSA (Huang et al., 2003), which accords with our research. In this study, a
complex crosstalk of phytohormones against pathogen infection in grape berries
were preliminarily profiled by dissecting endogenous phytohormones and their
correlative genes. Not only JA and ABA, but also C6-aldehydes and terpenes induced
by KODA application may be resistant substances against pathogen interaction in
grape berries.
Chapter 3 α-Ketol linolenic acid (KODA) application affects endogenous abscisic acid, jasmonic acid
and aroma volatiles in grapes infected by a pathogen (Glomerella cingulata)
70
In summary, KODA application may play a role of resistance against
pathogen infection in grape berries. ABA, JA, and some aroma volatiles may be
associated with the resistance against pathogenic infection in grapes treated by
KODA.
Chapter 4 General conclusion
71
CHAPTER 4
GENERAL CONCLUSION
Chapter 4 General conclusion
72
GENERAL CONCLUSION
In this study, the roles of JA and KODA that are the substances involved in
oxylipin pathway, on plant defense against fungal pathogen in grapes were
investigated. KODA may act a role in resistance to development of pathogen, which
was firstly demonstrated.
The level of endogenous JA was induced against to invasion of pathogen,
and showed higher concentration by the application of PDJ. The concentrations of
endogenous ABA also presented higher after inoculation and PDJ treatment, which
may be attributed in the increased expression of upstream enzyme gene VvNCED1
and downstream enzyme gene VvCYP707A1 in ABA biosynthetic pathway.
Synergistic and antagonistic effects have been reported in the interaction between the
ABA and JA signaling pathways (Moons et al., 1997; Staswick et al., 1992). A
cooperative function was exhibited between JA and ABA in our study. Aldehydes
and alcohols were mainly identified in large quantities during storage among all
groups, showing that volatile compounds in grapes predominantly consist of short
chain aldehydes and alcohols. (E)-2-hexenal was the most abundant volatile
compound identified in our study. It has been shown that t C6 aldehydes
((E)-2-hexenal and hexenal in our study) defensed against G. cingulate in grapes, in
keeping with previous report (Neri et al., 2006). Ester formation in grape berries
Chapter 4 General conclusion
73
needs a supply of substrate (alcohols) and the ester-forming enzyme, AAT (Wang
and Luca, 2005). As the ester concentration significantly increased in PDJ treatment
with inoculation, PDJ application might have influenced the accumulation of esters
by the stimulating activity of AAT. The terpenes or terpenoids constitute the largest
class of secondary products that appear to play important defensive roles in plants
(Gershenzon et al., 1992). A significant increase of terpenes was detected 6 days
after inoculation in PDJ with inoculation. Ethanol has been considered germistatic
against fruit and vegetable decay microorganisms (Penicillium digitatum and
Colletotrichum musae in an in vitro experiment) (Utama et al., 2002). The aspect of
aroma volatile emission in plant defense mechanism may be different among plants.
The effect of KODA application on defensing pathogen in grape was
investigated in chapter 3. The JA and ABA were stimulated and accumulated after
KODA treatment with inoculation, suggesting that the presence of a complex
network between JA and ABA for regulating the effects of resistance to pathogen,
following previous research (Adie et al., 2007). KODA application did not retard the
pathogen spreading on the agar medium, which suggests that KODA treatment may
increase the tolerant activity by inducing second metabolite against pathogen
infection in grape berries. KODA application induced accumulation of JA by
activating the VvLOX and VvAOS expression. Me-SA, which is a volatile ester
converted from SA, were higher in KODA+ inoc+ than those in KODA− inoc+ and the
Chapter 4 General conclusion
74
untreated control. ABA-mediated stress signals regulated SA biosynthesis by
inducing the SA biosynthetic enzyme gene SID2 in Arabidopsis (Seo and Park,
2010), which shows ABA signaling might act synergistically with SA signaling in
triggering plant immune response. Rojo et al. (2003) and Takahashi et al. (2004)
showed the antagonistic interaction between SA and JA signaling pathway. However,
Schenk et al. (2000) and Mur et al. (2006) revealed the synergetic action of SA and
JA in response to pathogens, which is in accordance with my result. Defense
responses involved in different signaling pathway are usually determined by
plant-pathogen interaction (Jones and Dangl, 2006). The interaction of KODA, JA,
and C6-aldehydes were involved in oxylipin pathway (Kishimoto et al., 2005).
Volatile C6 -aldehydes, JA were induced subjected to KODA application. JA and
C6-aldehydes are important in defense against microbial pathogen and insect attacks
(Li et al., 2005; Kishimoto et al., 2006). α-Farnesene is emitted by a number of plant
tissues in response to herbivory, wounding, pathogen and general defense, which
accords with this research (Huang et al., 2003). In my study, a complex crosstalk of
phytohormones against pathogen infection in grape berries were preliminarily
profiled by dissecting endogenous phytohormones and their correlative genes.
Summary
75
SUMMARY
The effects of the application of the jasmonic acid (JA) derivative n-propyl
dihydrojasmonate (PDJ) on lesion diameter, endogenous abscisic acid (ABA),
phaseic acid (PA), JA, aroma volatiles, and antioxidant activity were investigated in
grape berries infected by the pathogen Glomerella cingulata. The berries were
immersed in 0.4 mM PDJ solution before inoculation with the pathogen and stored at
25 °C for 12 days. Lesion diameters from the pathogen decreased upon PDJ
application. Endogenous ABA and JA concentrations increased in inoculated ‘Kyoho’
grape berries (Vitis labrusca × V. Vinifera). PDJ application before inoculation
increased ABA and PA concentrations, perhaps through the activation of VvNCED1
and VvCYP707A1 genes. The results suggest that the synergistic effect of JA and
ABA may play a role in the defence mechanism against pathogen infection in grape
berries. In addition, PDJ application generally increased the production of esters and
terpenes. These volatiles may also be associated with resistance to pathogen
infection.
Effects of α-ketol linolenic acid (KODA) application on endogenous ABA,
JA, and aroma volatiles were investigated in ‘Kyoho’ grape berries infected by a
pathogen (G. cingulata). The expressions of 9-cis epoxycaroteoid dioxigenase
(VvNCED1), ABA 8’-hydrixylase (VvCYP707A1), lipoxygenase (VvLOX), and allene
Summary
76
oxide synthase (VvAOS) were also examined. The grape berries were dipped in 0.1
mM KODA solution before inoculation with pathogen and stored at 25°C for 12 days.
The development of infection was significantly suppressed upon KODA treatment.
Endogenous ABA, JA, and phaseic acid (PA) were induced in inoculated berries.
KODA application before inoculation increased endogenous ABA, PA, and JA
through the activation of VvNCED1, VvCYP707A1, and VvAOS genes, respectively.
In addition, terpenes, methyl salicylate (Me-SA) and C6-aldehydes such as
(E)-2-hexenal and cis-3-hexenal associated with fungal resistance also increased in
KODA treated-fruit during storage. These results suggest that the synergistic effect
of JA, ABA, and some aroma volatiles induced by KODA application may be
responsible for the resistance against pathogen infection in grape berries.
Acknowlegement
77
ACKNOWLEGEMENT
It is a genuine pleasure to express my deepest sense of thanks and gratitude
to my advisor Professor Satoru Kondo. His timely advice, meticulous scrutiny and
scientific approach which helped me to a very great extent to accomplish my work. I
also owe a deep sense of gratitude to Professor Hitoshi Ohara, Senior Assistant
Professor Katsuya Ohkawa and Assistant professor Takanori Saito for their constant
inspirations, guidance, patience, encouragement and useful advices through my
research period. I am extremely thankful to Professor Masahiro Shishido, field of
biological environment in Chiba University, providing me valuable suggestions and
fungus applied in this research. I acknowledge with thanks the kindness of Assistant
Professor Hiromi Ikeura, Organization for the Strategic Coordination of Research
and Intellectual Properties in Meiji University, supporting extremely great extent
technical help in aroma volatiles analysis. I also extended my appreciation to
Professor Kazumitsu Miyoshi, field of Plant culture, Professor Masahiro Shishido
and Professor Hitoshi Ohara for their continuous guidance, comments, and
suggestions to complete this thesis.
I would like to express my thanks to all the members of Professor Kondo’s
Laboratory for their continuous encouragement, helpful cooperation, good friendship
and pleasant surroundings throughout the period of my doctor’s program.
Acknowlegement
78
Finally, I am very grateful for the love and support from my family and
friends, without their inspiration and dedication, this task would not have been
possible.
References
79
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