Download Effects of WClMV and root-flooding on white clover

Effects of WClMV and root-flooding on white clover
Maxime Gérard Corral
A thesis submitted for the degree of:
Master of Science
University of Otago
Dunedin, New Zealand
January 2013
Abstract
The legume white clover (Trifolium repens L.) is considered the most important forage
plant in New Zealand. It significantly contributes to increase the quality of pastures through
its ability to fix atmospheric nitrogen; providing high nutritional feed for livestock.
Trifolium repens is a common host for the Potexvirus White clover mosaic virus (WClMV),
the latter being readily mechanically transmitted via animal trampling and grazing, as well
as cropping practises. WClMV infection may strongly impact white clover’s performance,
hence affecting its ability to compete with other plants in the pasture. In the field, the
legume may also experience important stress induced by excess water in soils mainly due to
over-irrigation or heavy rainfall, a phenomenon likely to occur at higher frequency as a
consequence of climate change. When plants are subjected to biotic and abiotic stresses, the
outcome often involves the formation of reactive oxygen species (ROS) such as hydrogen
peroxide (H2O2), superoxide anion (O2•-) and hydroxyl radical (HO•). As highly reactive
species, ROS can damage the cell’s components, including DNA, proteins and membrane
lipids. To counteract the effect of ROS, plants have evolved non-enzymatic and enzymatic
antioxidant mechanisms that detoxify ROS and prevent cellular damage. The objective of
the current study was to investigate the effects of WClMV infection and root-flooding on
Trifolium repens’ performance, oxidative damage and the induction of antioxidants. These
experiments were conducted using two cultivars of white clover, cv. Huia and cv. Kopu II.
In response to both WClMV infection and root-flooding, white clover experienced
oxidative stress as evidenced by the accumulation of lipid peroxides and protein carbonyls.
Biomass accumulation, given as fresh weight and dry matter content, was affected by both
treatments but differently between varieties, cv. Kopu II being more sensitive than its
counterpart. Exposure to both stresses induced an increase in ascorbate and glutathione
levels, as well as enhancing the activity of the enzymatic antioxidants superoxide
dismutase, ascorbate peroxidase, glutathione peroxidase, catalase and glutathione reductase.
Glutathione S-transferase activity was not affected by both treatments. The levels of
reduced ascorbate and glutathione were unchanged in WClMV-infected plants, whereas the
root-flooding treatment significantly decreased their availability for the plant cell.
Interestingly, the interactive effect of Virus x Root-flooding was more damaging for the
ii
clovers, showing higher levels of oxidative stress and a down-regulation of antioxidants
when compared to uninfected/root-flooded plants. Under flooding treatment, white clover
developed aerenchymatous tissues in the roots, an important feature that may significantly
improve the legume’s tolerance towards submergence-induced stress. Furthermore, from
pasture feeding analyses, WClMV infection was shown to have no effect on the nutritional
composition of the clovers, in either cultivar.
Understanding how some biotic and abiotic factors can adversely affect the ability of
Trifolium repens to grow and compete with other plants in the field is an important aspect
for farmers, as it would provide valuable information for improving the quality of the
pastures in New Zealand.
iii
Résumé
Le trèfle blanc (Trifolium repens L.) est une espèce de légumineuse amplement utilisée en
Nouvelle Zélande en tant que plante fourragère. Du fait de son aptitude à fixer l’azote
atmosphérique, T. repens contribue grandement à enrichir la qualité des pâtures, fournissant
ainsi une alimentation à haute valeur nutritive pour le bétail. T. repens est également un
hôte fréquent du virus de la mosaïque du trèfle blanc (WClMV), ce dernier étant facilement
transmis par voie mécanique notamment par le biais du bétail (piétinement & pâturage) et
du matériel agricole pour l’entretien des pâtures. L’infection induite par WClMV peut avoir
de fortes incidences sur la performance du trèfle blanc, se résumant généralement par une
perte de capacité à entrer en compétition avec d’autres plantes pastorales. Au sein de son
environnement, la légumineuse peut également être soumise à des stress hydriques dus à un
excès d’eau dans les sols, les causes majeures étant sur-irrigation des terres agricoles ainsi
que fortes précipitations, un phénomène qui dans le futur à de grande chance de s’aggraver
en raison du changement climatique. Quand une plante se trouve sous la contrainte de stress
biotique ou abiotique, la formation d’espèces réactives de l’oxygène (ROS) tel que le
peroxyde d’hydrogène (H2O2), l’anion superoxyde (O2•-) et le radical hydroxyle (HO•), est
souvent mise en jeu. Hautement réactifs, les ROS peuvent facilement endommagées les
composantes de la cellule, notamment l’ADN, les protéines et les lipides membranaires.
Afin de se protéger des effets néfastes engendrés par les ROS et ainsi éviter des dommages
cellulaires, les plantes ont évoluées d’une manière à mettre en place des mécanismes antioxydatifs non-enzymatiques et enzymatiques. L’objectif de la présente thèse fut de mettre
en évidence les effets de l’infection par WClMV ainsi que de la submersion des racines sur
la performance de T. repens, les dégâts oxydatifs et l’induction d’antioxydants. Les
expériences ont été menées en utilisant deux cultivars de trèfle blanc, à savoir cv. Huia et
cv. Kopu II.
En réponse à l’infection par WClMV et au stress hydrique les plantes ont manifestées des
niveaux élevés de stress oxydatif, démontré par l’accumulation de peroxydes lipidiques et
de protéines carbonylées. La biomasse végétale, exprimée en termes de poids frais et de
contenu en matière sèche, fut affectée par les deux traitements mais de manière différente
selon les deux cultivars, cv. Kopu II ayant été plus sensible comparé à son partenaire.
iv
Les deux traitements ont également causés une augmentation des niveaux de glutathion et
d’ascorbate, ainsi qu’une forte induction de l’activité des enzymes superoxyde dismutase,
ascorbate peroxydase, glutathion peroxydase, catalase et glutathion reductase. En revanche,
l’activité enzymatique de la glutathion S-transferase n’a montré aucun changement. Les
proportions réduites d’ascorbate et glutathion sont restés identiques en réponse à l’infection
viral, tandis que le stress hydrique à significativement diminué leur disponibilité pour la
cellule végétale. Il est intéressant de noter que l’effet de l’interaction Virus x Stress
hydrique fut grandement néfaste pour les plantes, révélant des niveaux élevés de stress
oxydatifs ainsi qu’une sous-régulation des antioxydants en comparaison avec les plantes
submergées/non-infectées. Le trèfle blanc fut également capable de former des tissus
aérifères au niveau des racines quand ces dernières furent submergées, une caractéristique
importante pouvant améliorer la tolérance de la légumineuse envers de tels stress. De plus,
grâce à des analyses de nutrition, il a été démontré que WClMV n’eu aucun effet sur la
composition nutritionnelle de la plante.
Mettre en évidence la manière dont des facteurs biotiques et abiotiques peuvent
négativement affectés la capacité de T. repens à croître et entrer en compétition avec
d’autres espèces pastorales est un aspect important pour les agriculteurs. En effet, ceci
apporterait des informations de grande valeur pouvant aider à améliorer la qualité des
pâtures de Nouvelle Zélande.
v
Acknowledgements
I would like to express my deepest gratitude to my Masters supervisors Paul Guy and
David Burritt for their guidance and support throughout this project, and for providing
valuable technical support during the experiments. My appreciation also extends to all the
staff and students of the Botany Department for their kindness, help and support. I would
like to acknowledge Richard Easingwood, from the Department of Anatomy, for his
assistance in transmission electron microscopic observations of the viral particles. I am
really grateful to Damien Mather, from the Department of Marketing, who helped me with
the statistical analyses. Many thanks also go to all my friends and family for their support
and encouragement. Lastly and most importantly, I would like to thank my parents, Mireille
and Bernard Corral, who have always believed in me and supported me throughout my
academic path. Without them, this project would not have been possible. To them I
dedicate this thesis.
vi
Table of Contents
Abstract .................................................................................................................................. ii
Résumé .................................................................................................................................. iv
Acknowledgements ............................................................................................................... vi
List of Tables.......................................................................................................................... x
List of Figures ....................................................................................................................... xi
Abbreviations ....................................................................................................................... xv
1.
General introduction....................................................................................................... 1
1.1
1.1.1
Definition ......................................................................................................... 2
1.1.2
Transmission, infection process and movement of plant viruses ..................... 3
1.1.3
Effects of plant viruses on their hosts .............................................................. 5
1.2
Oxidative stress in plants ......................................................................................... 7
1.2.1
The importance of ROS in plant-pathogen interactions................................... 8
1.2.2
Non-enzymatic antioxidants .......................................................................... 10
1.2.3
ROS-scavenging enzymes.............................................................................. 11
1.2.4
Protein carbonylation & lipid peroxidation as markers of oxidative stress ... 14
1.3
Project introduction ............................................................................................... 17
1.3.1
White clover (Trifolium repens L.) ................................................................ 17
1.3.2
White clover mosaic virus: a foe for clover species ...................................... 21
1.4
2.
Insights and importance of plant viruses ................................................................. 1
Thesis objectives ................................................................................................... 23
Materials and Methods ................................................................................................. 25
2.1
Study organisms .................................................................................................... 25
2.1.1
WClMV inoculation ....................................................................................... 25
2.1.2
WClMV detection: DAS-ELISA protocol ..................................................... 26
vii
2.1.3
Observation of WClMV particles .................................................................. 26
2.1.4
Measurement of photosynthetic efficiency and growth ................................. 27
2.2
3.
2.2.1
Enzymatic antioxidants extraction and analysis ............................................ 28
2.2.2
Non-enzymatic antioxidants extraction and analysis ..................................... 30
2.2.3
Protein content and protein carbonyls extraction and analysis ...................... 31
2.2.4
Lipid peroxides extraction and analysis ......................................................... 31
2.3
Nutritional analysis ................................................................................................ 32
2.4
Root cross-sections ................................................................................................ 32
2.5
Statistical analysis ................................................................................................. 32
Antioxidant metabolism in White clover mosaic virus-infected Trifolium repens ..... 34
3.1
Introduction ........................................................................................................... 34
3.2
Methods ................................................................................................................. 34
3.3
Results ................................................................................................................... 36
3.3.1
Phenotypic variability in symptoms ............................................................... 36
3.3.2
Effect of WClMV infection on growth parameters........................................ 40
3.3.3
Markers of oxidative stress ............................................................................ 44
3.3.4
Non-enzymatic antioxidants .......................................................................... 46
3.3.5
Enzymatic antioxidants .................................................................................. 50
3.3.6
Nutrient analyses ............................................................................................ 57
3.4
4.
Biochemical assays ................................................................................................ 28
Discussion ............................................................................................................. 62
The effect of root-flooding on oxidative damage and antioxidant metabolism in
Trifolium repens infected or not with WClMV ................................................................... 71
4.1
Soil flooding: an important abiotic stress .............................................................. 71
4.2
Methods ................................................................................................................. 77
4.3
Results ................................................................................................................... 79
viii
4.3.1
Effect of root-flooding on the root system ..................................................... 79
4.3.2
Effect of WClMV & root-flooding on growth parameters ............................ 82
4.3.3
Markers of oxidative stress ............................................................................ 86
4.3.4
Non-enzymatic antioxidants .......................................................................... 90
4.3.5
Enzymatic antioxidants .................................................................................. 98
4.4
5.
Discussion ........................................................................................................... 109
General discussion ..................................................................................................... 115
5.1
Overview of the study ......................................................................................... 115
5.2
Future research .................................................................................................... 118
5.3
Conclusions ......................................................................................................... 119
ix
List of Tables
Table 1
Summary of methods for Pasture Feed Quality Analysis ................................. 33
Table 2
% Dry matter in uninfected and WClMV-infected T. repens ........................... 42
Table 3
Summary of ANOVA results for growth parameters, oxidative damage and
antioxidant activities measured in Experiment 1 .............................................. 56
Table 4
Content of nitrogen and ash in T. repens Huia & Kopu II at each harvest ....... 57
Table 5
Content of crude protein (CP), acid detergent fibre (ADF) and neutral detergent
fibre (NDF) in T. repens Huia & Kopu II at each harvest ................................ 58
Table 6
Content of soluble sugars and starch in T. repens Huia & Kopu II at each
harvest ............................................................................................................... 59
Table 7
Digestible organic matter in the dry matter (DOMD) and metabolisable energy
(ME) in T. repens Huia & Kopu II at each harvest ........................................... 60
Table 8
Summary of ANOVA results for nutritional parameters measured in
Experiment 2 ..................................................................................................... 61
Table 9
Summary of ANOVA results for growth parameters, oxidative damage and
antioxidant activities measured in Experiment 2 ............................................ 108
x
List of Figures
Figure 1
Model of TMV-like transport of viral RNA through a plasmodesma................. 4
Figure 2
Model of production of different reactive oxygen species deriving from ground
state oxygen ......................................................................................................... 9
Figure 3
Interactions between ROS and scavenging system ........................................... 16
Figure 4
Stolon characteristics of white clover ............................................................... 18
Figure 5
Pictures showing leaves and inflorescences of white clover............................. 19
Figure 6
Root from white clover showing nodules ......................................................... 20
Figure 7
Observation of rod-shaped virus particles of White clover mosaic virus with a
transmission electron microscope at 38,000x magnification ............................ 22
Figure 8
Organisation of WClMV genomic RNA ........................................................... 22
Figure 9
Leaf symptoms induced by WClMV ................................................................ 24
Figure 10 Experimental design showing white clover Huia and Kopu II ......................... 35
Figure 11 Timeline showing PAM measurements and harvests of Experiment 1 ............ 36
Figure 12 Phenotypic variability in uninfected and WClMV-infected leaves of white
clover Huia and Kopu II .................................................................................... 37
Figure 13 Cultivars Huia and Kopu II displaying phenotypic differences between
treatments (i.e. Uninfected vs Infected) at 3 wpi............................................... 38
Figure 14 Cultivars Huia and Kopu II displaying lessened phenotypic differences between
treatments (i.e. Uninfected vs Infected) at 9 wpi............................................... 39
Figure 15 Mean above-ground fresh weight of uninfected and WClMV-infected white
clover Huia and Kopu II at each harvest ........................................................... 40
Figure 16 Mean above-ground fresh weight of white clover Huia and Kopu II within
treatments at each harvest ................................................................................. 41
Figure 17 Dry matter content of above-ground biomass of uninfected and WClMVinfected white clover Huia and Kopu II at each harvest ................................... 42
Figure 18 Effect of WClMV on photosynthetic efficiency in Huia and Kopu II .............. 43
Figure 19 Mean protein carbonyl content in uninfected and WClMV-infected Huia and
Kopu II at each harvest...................................................................................... 44
Figure 20 Levels of lipid peroxide in uninfected and WClMV-infected Huia and Kopu II
at each harvest ................................................................................................... 45
xi
Figure 21 Total ascorbate in uninfected and WClMV-infected Huia and Kopu II at each
harvest ............................................................................................................... 46
Figure 22 % Reduced ascorbate in uninfected and WClMV-infected Huia and Kopu II at
each harvest ....................................................................................................... 47
Figure 23 Total glutathione in uninfected and WClMV-infected Huia and Kopu II at each
harvest.. ............................................................................................................. 48
Figure 24 % Reduced glutathione in uninfected and WClMV-infected Huia and Kopu II at
each harvest ....................................................................................................... 49
Figure 25 Mean superoxide dismutase activity in uninfected and WClMV-infected Huia
and Kopu II at each harvest ............................................................................... 50
Figure 26 Mean ascorbate peroxidase activity in uninfected and WClMV-infected Huia
and Kopu II at each harvest ............................................................................... 51
Figure 27 Mean glutathione peroxidase activity in uninfected and WClMV-infected Huia
and Kopu II at each harvest ............................................................................... 52
Figure 28 Mean catalase activity in uninfected and WClMV-infected Huia and Kopu II at
each harvest ....................................................................................................... 53
Figure 29 Mean glutathione S-transferase activity in uninfected and WClMV-infected
Huia and Kopu II at each harvest ...................................................................... 54
Figure 30 Mean glutathione reductase activity in uninfected and WClMV-infected Huia
and Kopu II at each harvest ............................................................................... 55
Figure 31 General overview of metabolic pathways and ROS generation under hypoxic
conditions .......................................................................................................... 76
Figure 32 Experimental design showing white clover Huia and Kopu II placed on a waterfilled bench ........................................................................................................ 78
Figure 33 Timeline showing PAM measurements and harvests of Experiment 2 ............ 78
Figure 34 Comparison of root systems between a control and a root-flooded plant ......... 79
Figure 35 Cross-sections of non-waterlogged and waterlogged roots stained with toluidine
blue. ................................................................................................................... 80
Figure 36 Root-flooded plants showing root formation in the water ................................ 81
Figure 37 Above-ground fresh weight of uninfected, WClMV-infected, uninfected/rootflooded and WClMV-infected/root-flooded white clover Huia ........................ 82
xii
Figure 38 Above-ground fresh weight of uninfected, WClMV-infected, uninfected/rootflooded and WClMV-infected/root-flooded white clover Kopu II ................... 83
Figure 39 Dry matter content of above-ground biomass of uninfected, WClMV-infected,
uninfected/root-flooded and WClMV-infected/root-flooded white clover Huia
and Kopu II........................................................................................................ 84
Figure 40 Maximal photochemical efficiency (Fv/Fm) of uninfected, WClMV-infected,
uninfected/root-flooded and WClMV-infected/root-flooded white clover Huia
and Kopu II........................................................................................................ 85
Figure 41 Protein carbonyl content in uninfected, WClMV-infected, uninfected/rootflooded and WClMV-infected/root-flooded Huia plants at each harvest ......... 86
Figure 42 Protein carbonyl content in uninfected, WClMV-infected, uninfected/rootflooded and WClMV-infected/root-flooded Kopu II plants at each harvest ..... 87
Figure 43 Lipid peroxide content in uninfected, WClMV-infected, uninfected/rootflooded and WClMV-infected/root-flooded Huia plants at each harvest ......... 88
Figure 44 Lipid peroxide content in uninfected, WClMV-infected, uninfected/rootflooded and WClMV-infected/root-flooded Kopu II plants at each harvest ..... 89
Figure 45 Total ascorbate concentration in uninfected, WClMV-infected, uninfected/rootflooded and WClMV-infected/root-flooded Huia plants at each harvest ......... 90
Figure 46 Total ascorbate concentration in uninfected, WClMV-infected, uninfected/rootflooded and WClMV-infected/root-flooded Kopu II plants at each harvest ..... 91
Figure 47 % Reduced ascorbate in uninfected, WClMV-infected, uninfected/root-flooded
and WClMV-infected/root-flooded Huia plants at each harvest ....................... 92
Figure 48 % Reduced ascorbate in uninfected , WClMV-infected, uninfected/root-flooded
and WClMV-infected/root-flooded Kopu II plants at each harvest .................. 93
Figure 49 Total
glutathione
concentration
in
uninfected,
WClMV-infected,
uninfected/root-flooded and WClMV-infected/root-flooded Huia plants at each
harvest ............................................................................................................... 94
Figure 50 Total
glutathione
concentration
in
uninfected,
WClMV-infected,
uninfected/root-flooded and WClMV-infected/root-flooded Kopu II plants at
each harvest ....................................................................................................... 95
Figure 51 % Reduced glutathione in uninfected, WClMV-infected, uninfected/rootflooded and WClMV-infected/root-flooded Huia plants at each harvest ......... 96
xiii
Figure 52 % Reduced glutathione in uninfected, WClMV-infected, uninfected/rootflooded and WClMV-infected/root-flooded Kopu II plants at each harvest ..... 97
Figure 53 Superoxide dismutase activity in uninfected, WClMV-infected, uninfected/rootflooded and WClMV-infected/root-flooded Huia plants at each harvest ......... 98
Figure 54 Superoxide dismutase activity in uninfected, WClMV-infected, uninfected/rootflooded and WClMV-infected/root-flooded Kopu II plants at each harvest ..... 99
Figure 55 Ascorbate peroxidase activity in uninfected, WClMV-infected, uninfected/rootflooded and WClMV-infected/root-flooded Huia plants at each harvest ....... 100
Figure 56 Ascorbate peroxidase activity in uninfected, WClMV-infected, uninfected/rootflooded and WClMV-infected/root-flooded Kopu II plants at each harvest ... 101
Figure 57 Glutathione
peroxidase
activity
in
uninfected,
WClMV-infected,
uninfected/root-flooded and WClMV-infected/root-flooded Huia plants at each
harvest ............................................................................................................. 102
Figure 58 Glutathione
peroxidase
activity
in
uninfected,
WClMV-infected,
uninfected/root-flooded and WClMV-infected/root-flooded Kopu II plants at
each harvest ..................................................................................................... 103
Figure 59 Catalase activity in uninfected, WClMV-infected, uninfected/root-flooded and
WClMV-infected/root-flooded Huia plants at each harvest ........................... 104
Figure 60 Catalase activity in uninfected, WClMV-infected, uninfected/root-flooded and
WClMV-infected/root-flooded Kopu II plants at each harvest ....................... 105
Figure 61 Glutathione reductase activity in uninfected, WClMV-infected, uninfected/rootflooded and WClMV-infected/root-flooded Huia plants at each harvest ....... 106
Figure 62 Glutathione reductase activity in uninfected, WClMV-infected, uninfected/rootflooded and WClMV-infected/root-flooded Kopu II plants at each harvest ... 107
Figure 63 Schematic representation of the levels of oxidative damage and antioxidants
that were significantly affected by root-flooding in virus-free and WClMVinfected T. repens ............................................................................................ 112
xiv
Abbreviations
ACC
1-amino cyclopropane 1-carboxylic acid
ADF
Acid detergent fibre
ADH
Alcohol dehydrogenase
AMV
Alfalfa mosaic virus
ANOVA
Analysis of variance
ANPs
Anaerobic proteins
APX
Ascorbate peroxidase
ATP
Adenosine triphosphate
Avr genes
Avirulence genes
BWYV
Beet western yellows virus
BYDV
Barley yellow dwarf virus
BYMV
Bean yellow mosaic virus
CAT
Catalase
CfMV
Cocksfoot mottle virus
CMV
Cucumber mosaic virus
CMV[Y]
Cucumber mosaic virus strain Y
CP
Coat protein
CP(2)
Crude protein
cv.
Cultivar
CYVV
Clover yellow vein virus
DAS-ELISA Double antibody sandwich-enzyme linked immunosorbent assay
DHA
Dehydroascorbate
DHAR
Dehydroascorbate reductase
DMC
Dry matter content
DOMD
Digestible organic matter in the dry matter
dsRNA
Double-stranded RNA
EpYVV
Eupatorium yellow vein virus
Fv/Fm
Maximum photochemical efficiency
FW
Fresh weight
GLM
Generalized linear model
xv
GPX
Glutathione peroxidase
GR
Glutathione reductase
GSH
Glutathione
GSSG
Glutathione disulphide (oxidized glutathione)
GST
Glutathione S-transferase
H2O2
Hydrogen peroxide
HR
Hypersensitive response
HSP
Heat shock protein
LALV
Lucerne Australian latent virus
LDH
Lactate dehydrogenase
MDA
Monodehydroascorbate
MDAR
Monodehydroascorbate reductase
ME
Metabolisable energy
MP
Movement protein
N2
Atmospheric nitrogen or dinitrogen
NDF
Neutral detergent fibre
O2
Oxygen
O2•-
Superoxide
1
O2
Singlet oxygen
3
O2
Triplet oxygen
OEC
Oxygen-evolving complex
OH•
Hydroxyl radical
ORF
Open reading frame
PaMMoV
Paprika mild mottle virus
PAR
Photosynthetically active radiation
PDC
Pyruvate decarboxylase
PLRV
Potato leafroll virus
PMMoV
Pepper mild mottle virus
PPV
Plum pox virus
PR
Pathogenesis-related
PSII
Photosystem II
PUFAs
Polyunsaturated fatty acids
xvi
PVX
Potato virus X
PVY
Potato virus Y
NTN
PVY
Potato virus YNTN
QTLs
Quantitative trait loci
R genes
Resistance genes
RCNMV
Red clover necrotic mosaic virus
RCVMV
Red clover vein mosaic virus
ROS
Reactive oxygen species
RYMV
Rice yellow mottle virus
Rubisco
Ribulose biphosphate carboxylase-oxygenase
SA
Salicylic acid
SAR
Systemic acquired resistance
SbDV
Soybean dwarf virus
ScYLV
Sugarcane yellow leaf virus
SD
Standard deviation
SE
Standard error of the mean
SEL
Size exclusion limit
siRNAs
Small interfering RNAs
SOD
Superoxide dismutase
ssRNA
Single-stranded RNA
TGB
Triple gene block
TMV
Tobacco mosaic virus
ToYMV
Tomato yellow mosaic virus
TSWV
Tomato spotted wilt virus
vRNA
Viral RNA
WClMV
White clover mosaic virus
WMV
Watermelon mosaic virus
ZYMV
Zucchini yellow mosaic virus
xvii
1. General introduction
“Perhaps it does frost
In this village morn by morn
For the grass I saw in the field of summertime
Has already turned yellow”
Written in 752 AD by Koken, empress of Japan, this poem is deemed to be the earliest
record describing plant virus disease. The term “grass” mentioned here refers to
Eupatorium plants (Asteraceae) displaying vein-yellowing symptoms on their leaves,
known nowadays to be caused by the geminivirus Eupatorium yellow-vein virus (EpYVV)
along with a DNA satellite (Noctor et al., 2012; Saunders et al., 2003).
Plant molecular virology initially arose from the study of the well-known Tobacco mosaic
virus (TMV) in the early 20th century. Symptoms induced by TMV were described decades
earlier in Colombia, where tobacco crops infected with an unknown disease manifested the
development of light-dark green mosaic patterns on the leaves. In 1879, German Adolph
Mayer first referred to the disease as “the mosaic disease of tobacco”. A few years later, he
reported that the disease was readily transmissible to uninfected plants by simply rubbing
their leaves with infected leaf sap (Scholthof, 2008). In 1892, Russian botanist Dmitri
Ivanovsky showed that sap from tobacco plants infected by the same disease conserved its
infectious properties after passing through a bacteria-proof filter candle. At that time, the
causative agent was thought to be a toxin (Hull, 2009). However, microbiologist Martinus
W. Beijerinck (1898) carried out similar experiments and showed that the infectious
particle was able to diffuse through agar and multiply in infected tissue. He first named it
contagium vivium fluidum (i.e. contagious living fluid) and used the term “virus” to
discriminate it from bacteria (Hull, 2009; Scholthof, 2008).
1.1 Insights and importance of plant viruses
Plant viruses are regarded as economically important pathogens infecting plants worldwide
and causing a broad range of diseases associated with substantial financial losses (Hull,
2009; Rodoni, 2009). For instance, virus-induced diseases have caused estimated annual
1
losses of $1.5 billion in rice in South-East Asia, over $20 million in potato production in
the United Kingdom (Andret-Link and Fuchs, 2005), and up to $63 million in apples in the
United-States (Cembali et al., 2003). Apart from conspicuous effects on crop yield and
quality of products, viral diseases may also (1) increase sensitivity of plants to
environmental stresses (e.g. drought, frost, attacks by other pathogens), (2) increase costs of
means used to limit disease and maintain crop health, and (3) reduce the value of crop
products at market (Hull, 2009). Therefore, in the challenge of limiting the negative
impacts of viruses on crops and improving yields, the study of plant-virus interactions is of
great importance. It should also be mentioned that viral infection may have positive
outcomes on plant survival under certain conditions, such as improving drought tolerance
(Xu et al., 2008), increasing sugar content in leaves (Gonçalves et al., 2005; Herbers et al.,
1997), and affecting the behaviour of insect vectors (e.g. attraction and arrest of the vector)
(Eigenbrode et al., 2002), to cite a few.
1.1.1
Definition
Viruses are small obligate parasites entirely relying on their host cells. They utilize the
cellular machinery of their hosts in order to replicate and move from cell to cell (Gergerich
and Dolja, 2006). A virus’ nucleic acid may be single- or double-stranded, DNA or RNA,
and is often prone to high genetic variability by means of point mutations, recombination
and reassortment events (Matthews and Hull, 2002; Roossinck, 1997). Although the singlestranded RNA (ssRNA) viruses were thought to represent the most common group of plant
viruses (Carr, 2007), current research has unveiled many new species of Geminiviruses
(ssDNA viruses), making the latter the largest family of plant-infecting viruses known to
date (personal communication). Because plant viruses have to cope with extra-cellular
barriers, i.e. cell walls, and pass from infected to adjacent cells to spread within the host,
having small genomes is likely to counteract these constraints (Desbiez et al., 2011;
Madigan et al., 2003). The structure of plant viruses is relatively simple. The viral genome
is surrounded and protected by a protein shell called capsid. The latter is composed of
structural monomeric units, or capsomers, arranged in such a way that they shape the viral
particle either in helical or icosahedral symmetry. Furthermore, a few plant viruses possess
an extra envelope of lipoprotein with glycoproteins anchored to it (Nayudu, 2008).
2
1.1.2
Transmission, infection process and movement of plant viruses
Plant viruses are mostly transmitted from host to host via biological vectors, such as
arthropods (the predominant virus-transmitting vectors in plants), nematodes, and fungi.
The most common insects involved in plant virus propagation are aphids, with more than
200 species identified as efficient vectors (Andret-Link and Fuchs, 2005; Roossinck, 1997).
It is interesting to note that some viruses are capable of proliferating in both plant and
insect hosts. This has led to the hypothesis that some plant viruses might have originated
from insect viruses (Desbiez et al., 2011). Transmission can also occur through human
activities, such as grafting and vegetative propagation, cropping techniques and breeding
(e.g. wounds caused by mechanical damage, including animal trampling or cropping
materials, can facilitate virus transmission), and via the introduction of pre-infected crops,
among others. In some cases, plant viruses are propagated through seed and pollen
produced and released by infected plants (i.e. vertical transmission) (Andret-Link and
Fuchs, 2005; Matthews and Hull, 2002), a mechanism used by over 20% of plant viruses
(Amari et al., 2009) and this number is still growing.
After entering the host cell, the viral particle releases its genome from the capsid, a process
called uncoating. The genomic RNA (or DNA) is subsequently translated and replicated by
using the cell machinery to form new virions. Replication of RNA viruses requires the
activity of replicase complexes. The latter are constituted of both viral components, such as
the RNA-dependent RNA polymerase and other replication-involved viral proteins, and
various host factors. The process of viral replication is costly for the infected organism as it
uses host proteins and hijacks different cellular pathways for its own profit (Nagy and
Pogany, 2011; Nicaise and Revers, 2001).
A critical step of the infection is the ability of the virus to spread within the plant.
Movement of plant viruses occurs in two phases: a cell-to-cell movement, followed by a
long-distance movement (Rao and Choi, 2002). Transport into neighbouring cells is
accomplished by passing through the plasmodesmata, i.e. specialized intercellular structure
forming connections and cytoplasm continuity between adjacent cells, thus enabling
communication and transport of molecules (Burch-Smith and Zambryski, 2012; Gergerich
3
and Dolja, 2006). A number of plant viruses encode specific proteins, called movement
proteins (MPs), of which the main function is to alter plasmodesmatal channels in order to
facilitate the passage of the pathogen. While some viruses only require a single MP, others
necessitate further viral proteins to spread effectively (Gergerich and Dolja, 2006;
Taliansky et al., 2008). One well-studied viral MP is P30, a protein encoded by the ssRNA
of TMV. P30 is thought to act as a molecular chaperone for the viral genome, a function
inferred by its capacity to bind to single-stranded nucleic acids. By interacting with the
viral RNA, P30 would be able to change its conformation into a long, unfolded and thin
protein-nucleic acid complex, therefore facilitating its transport (Citovsky and Zambryski,
1991). Likewise, some viral MPs have been shown to interact with some elements of the
cytoskeleton in order to optimize the movement of virions towards the plasmodesmata (Fig.
1). The last step of the infection is achieved when the pathogen has crossed through the
different cellular compartments and is loaded into the sieve elements, thereby spreading
systemically within the plant (Carrington et al., 1996).
Microtubule
Microfilament
MPs-associated
vRNA
Proteins
Cytoplasm
Desmotubule
Plasma membrane
Cell wall
Cytoplasmic
sleeve
Endoplasmic
reticulum
Figure 1 Model of TMV-like transport of viral RNA (vRNA) through a plasmodesma. Structurally,
plasmodesmata are composed of plasma membrane, forming the outer layer of the channel, and of a
compacted portion of endoplasmic reticulum in the axial centre, or desmotubule. Membrane-associated
proteins are located within the plasmodesmatal channel and are likely to regulate molecular traffic. Cell-tocell transport mainly occurs in the cytoplasmic sleeve, i.e. the space between the plasma membrane and the
desmotubule (Burch-Smith and Zambryski, 2012; Carrington et al., 1996).
4
1.1.3
Effects of plant viruses on their hosts
The impacts of viral infection on plant metabolism and development are diverse and vary
greatly among different, or even similar, strains of viruses (Reinero and Beachy, 1989).
Alteration in host gene expression is one of the numerous consequences attributed to
viruses. The so-called heat shock proteins (HSPs)-encoding genes and defence genes, such
as the pathogenesis-related (PR) genes, are often expressed as a marker of virus-induced
cellular stress response (Whitham et al., 2006). The responses observed in incompatible
interactions, i.e. when resistance to a specific pathogen occurs through the induction of
plant resistance (R) genes (Soosaar et al., 2005), often involve increased levels of salicylic
acid (SA) acting as a mediator for the expression of PR genes. Although high levels of SA
are not seen in compatible interactions, in which the plant host is described as susceptible to
the infectious agent, expression of defence-related genes may be triggered by basal SA
levels. It is well accepted that perturbation in both synthesis and regulation of plant
hormones is a common consequence of viral infection and may significantly impact growth
and development of the host (Soosaar et al., 2005; Whitham et al., 2006). However,
enhanced synthesis of phytohormones may play an important role in plant resistance, as
shown in resistant potato cultivars infected with Potato virus YNTN (PVYNTN) displaying
increased concentration of jasmonic acid post-infection (Kovac et al., 2009).
Changes in protein synthesis and regulation have also been observed under viral infection.
Virus-induced mosaic and yellowing diseases are often linked to a dramatic decrease in
ribulose bisphosphate carboxylase-oxygenase (Rubisco) content. Likewise, as exemplified
by TMV during its replication stage, up to 75% of the host’s protein synthesis might be
impeded. It is thought that disruption in protein regulation mainly occurs at the translational
level (Hull, 2009).
In RNA-virus infected cells, membrane and organelle structures are usually altered. These
modifications may lead to the formation of spherules and/or (multi)vesicular structures
often attached to organelles or other components of the cytosol. Known as virus factories,
viroplasms, virosomes or virus inclusions, these pathogen-induced sub-compartments are
5
used for viral replication and may be involved in translation and cell-to-cell movement of
the viral RNA. Virus inclusions may also prevent recognition of viral RNA by the cell,
along with the activation of plant defence mechanisms (Laliberté and Sanfaçon, 2010).
Viruses are also able to affect photosynthesis of infected plants. It generally results in
decreased carbon fixation, a feature shared by mosaic and yellowing diseases, as well as in
the development of leaf symptoms such as chlorosis and necrosis (Hull, 2009). This is often
due to disruption of chloroplast structure and function. For instance, Nicotiana
benthamiana L. leaves infected with Pepper and Paprika mild mottle viruses (PPMoV &
PAMMoV, respectively) show decreased photosynthetic electron transport in photosystem
II (PSII) (Rahoutei et al., 2000). Likewise, accumulation of viral coat protein (CP) in the
chloroplasts of tobacco leaves (Nicotiana tabacum L.) infected with an aggressive
chlorosis-inducing strain of TMV was shown to be related to reduced electron flow within
PSII (Reinero and Beachy, 1989). The inhibitory effect of CP on PSII activity was
speculated to be the result of CP binding to protein components of PSII, therefore altering
their functions. Furthermore, in tobacco plants infected with Cucumber mosaic virus strain
Y (CMV[Y]), PSII inhibition was correlated with a down-regulation of the polypeptides
constituting the oxygen-evolving complex (OEC) (Takahashi and Ehara, 1992).
It is also widely accepted that viruses have strong effects on respiration, usually defined as
net oxygen uptake. As reported by Leal & Lastra (1984), the respiration rate of Tomato
yellow mosaic virus (ToYMV)-infected tomato leaves was 80-100% higher than that for
healthy plants. Although increased respiration rate is a common feature observed in many
host-virus combinations, it is often lower in chronically infected leaves than in healthy ones
(Hull, 2009).
A number of studies have shown that plant viruses were also able to influence carbohydrate
metabolism. The effects of Cucumber mosaic virus (CMV) on carbon assimilation and
transport were investigated by Shalitin & Wolf (2000) in melon plants. The infection
induced increased concentrations of fructose and glucose, and a significant reduction in
starch content in infected leaves. Similarly, Livingston et al. (1998) reported that Barley
yellow dwarf virus (BYDV) affected carbohydrate concentrations in cereals, and increased
6
leaf carbohydrate content was shown in Sugarcane yellow leaf virus (ScYLV)-infected
sugarcane plants (Gonçalves et al., 2005). Likewise, transgenic tobacco plants expressing
Potato leafroll virus (PLRV) movement protein exhibited high levels of soluble sugars and
starch. Accumulation of carbohydrates in infected leaves can result in decreased
photosynthetic efficiency and rubisco activity, a consequence attributed to reduced
photosynthesis-related gene expression. The latter mechanism has been suggested to occur
through photosynthate-mediated feed-back inhibition (Herbers et al., 1997). Besides
marked effects on primary metabolism that may affect growth and development of their
hosts, viruses may induce changes in plant secondary metabolites that are often involved in
defence mechanisms (Berger et al., 2007).
1.2 Oxidative stress in plants
In plant-virus interactions, when a plant is unable to specifically recognize a given
pathogen, the interaction is referred to as compatible. In this instance, the virus is virulent
and the plant is susceptible. In contrast, when the virus is efficiently identified as pathogen
by the host, the established interaction is called incompatible, in which the virus is termed
avirulent and the plant is resistant (Stange, 2006). Described by Flor (1971), the gene-forgene theory suggests that, in incompatible interactions, the products of both plant resistance
(R) genes and pathogenic avirulence (avr) genes specifically interact with each other, in a
direct or indirect fashion, to eventually lead to the induction of the hypersensitive response
(HR) (Dardick and Culver, 1999). When the HR is triggered, it occurs at the infection site
and results in programmed cell death, thus limiting the pathogen spread to surrounding
cells (Stange, 2006).
In both incompatible and compatible interactions, one of the earliest responses to the
pathogen attack is the accumulation of reactive oxygen species (ROS; such as superoxide,
hydrogen peroxide & hydroxyl radical), a process named the “oxidative burst” (Hakmaoui
et al., 2012). ROS play a number of functions in the establishment of plant defence,
including cell-wall strengthening through the cross-linking of glycoproteins, regulation of
intracellular transduction signals, and enhanced systemic acquired resistance (SAR)
(Mandal et al., 2011; Yoshioka et al., 2008). An important role of ROS generation upon
7
pathogen recognition is the induction of the HR. However, in some plant-pathogen
interactions, ROS-producing oxidative burst and HR are not always correlated and may
even be antagonistic (Torres, 2010).
1.2.1
The importance of ROS in plant-pathogen interactions
In plants, ROS are naturally formed as by-products of oxygen-dependent metabolism.
Respiration and photosynthesis, two crucial aerobic metabolic processes, induce the
production of ROS in organelles such as mitochondria, chloroplasts and peroxisomes (Apel
and Hirt, 2004; Torres et al., 2006). The electron transport chains within chloroplasts and
mitochondria are the main source of ROS. Moreover, during light-dependent reactions,
excited chlorophyll may also induce the formation of ROS via direct energy transfer to
oxygen (Rinalducci et al., 2008). Accumulation of ROS may also be caused by a variety of
biotic and abiotic stresses (e.g. pathogen invasion, UV radiation, salt stress,
photorespiration, drought). As highly reactive molecules, ROS can damage DNA, proteins
and lipids (Apel and Hirt, 2004; Torres et al., 2006).
Upon pathogen recognition, the oxidative burst gives rise to the generation of ROS in the
apoplast and within the plant cell. Apoplastic production of ROS is mainly mediated by
plasma membrane-bound NADPH oxidases, or respiratory burst oxidases, as well as
through the activity of cell wall peroxidases (Torres, 2010). Following infection, ROS
accumulation often occurs in a biphasic manner: a primary low-amplitude production
followed by, in incompatible interactions, a sustained production of ROS that triggers
specific host resistance (Torres et al., 2006). Indeed, for many plant-pathogen interactions,
resistant plants have been shown to produce higher levels of hydrogen peroxide (H2O2)
than susceptible plants (Mandal et al., 2011).
Despite the high toxicity associated with ROS, plants have evolved ways of using such
reactive agents as signalling molecules (Rinalducci et al., 2008). In plant-microorganism
interactions, ROS play a plethora of roles by acting in a wide range of responses. They are
involved in cell wall strengthening, and also induce defence mechanisms by modulating
gene expression through modification of cellular redox status or interaction with other
8
cellular components (Apel and Hirt, 2004). ROS might work in conjunction with
phytohormones such as salicylic acid, ethylene and nitric oxide in specific signalling
pathways. These complex and multiple interactions between ROS and other signalling
molecules give rise to a variety of responses triggered during pathogen-induced stress
(Torres, 2010; Torres et al., 2006).
Different processes lead to the production of ROS. Molecular oxygen in its normal ground
state, or non-excited state, is referred to as triplet oxygen (3O2). Through direct energy
transfer, triplet oxygen may be converted to a toxic and higher energy state of oxygen, or
singlet oxygen (1O2). Furthermore, sequential reduction of molecular oxygen leads to the
generation of superoxide (O2•-), H2O2 and hydroxyl radical (OH•). In the NADPHdependent oxidase system, O2•- production is facilitated by the reduction of oxygen, with
NADPH acting as an electron donor. Other candidates, such as cell wall peroxidases, may
enhance H2O2 and OH• accumulation when using adequate reductants (Fig. 2) (Apel and
Hirt, 2004).
3O
2+
e-
O2•- Superoxide
O2•- + e- + 2H+
1O
H2O2 Hydrogen peroxide
H2O2 + e- + H+
2
Singlet
oxygen
H2O + OH• Hydroxyl radical
OH• + e- + H+
H2O Water
Figure 2 Model of production of different reactive oxygen species deriving from ground state oxygen (Apel
and Hirt, 2004; Madigan et al., 2003).
In plant cells, the balance between the formation and removal of ROS must be accurately
regulated. However, this equilibrium might be disturbed by various biotic and abiotic
stresses. In order to efficiently remove excess ROS, plants have evolved ROS scavenging
mechanisms using non-enzymatic and enzymatic antioxidants (Apel and Hirt, 2004; Torres,
2010; Torres et al., 2006).
9
1.2.2
Non-enzymatic antioxidants
The low molecular weight ascorbate, glutathione (GSH, its reduced form) and α-tocopherol
are the major non-enzymatic antioxidants found in plants and play central roles in
maintaining cell homeostasis by acting as cellular redox buffers. Other non-enzymatic
components such as flavonoids, alkaloids and carotenoids have also been characterised as
ROS detoxifiers (Apel and Hirt, 2004). In the current study, only the levels of ascorbate and
GSH were quantified.
In plants, the tripeptide glutathione, or γ-glutamylcysteinyl-Gly, is the most common source
of non-protein thiol. It acts in a broad range of biological processes (Edwards et al., 1994;
Xiang et al., 2001). As a sulphur-containing molecule, GSH plays key roles in sulphur
assimilation and is involved in the formation of nitrogen-fixing nodules in legumes. In
response to pathogen attacks, levels of glutathione appear to be a decisive factor as it has
been associated with enhanced expression of defence genes and synthesis of hormones and
secondary metabolites. Changes in GSH redox status following infection may also be
linked to HR, although this assumption has yet to be confirmed (Noctor et al., 2012).
Likewise, glutathione is thought to protect plants from a variety of environmental stresses.
For instance, transgenic Arabidopsis plants with low levels of GSH display high sensitivity
to heavy metals, as inferred from the phytochelatin property of polymerized GSH (Xiang et
al., 2001). In virus-infected plants, elevated glutathione content has been found to be
related to resistance. Zucchini yellow mosaic virus (ZYMV)-infected Cucurbita pepo plants
with artificially elevated glutathione levels showed decreased or suppressed virus-induced
symptoms, whereas control ZYMV-inoculated plants had reduced GSH contents along with
development of symptoms (Zechmann et al., 2007).
As a major plant metabolite, L-ascorbic acid or ascorbate (anion form) seems to occur in all
photosynthetic organisms. In plants, under non-stressful conditions, up to 90% of the
ascorbate pool can be present in the reduced form. Ascorbate is a powerful antioxidant that
reacts readily with ozone, superoxide, singlet oxygen and hydrogen peroxide (Conklin,
2001; Smirnoff, 1996). It also acts in many other processes, such as cell wall growth and
cell expansion, photosynthesis and photoprotection, and synthesis of phytohormones. In the
10
cell, ascorbate occurs in all compartments, including the apoplast, cytoplasm and organelles
(Smirnoff and Wheeler, 2000). During pathogen infection, levels of ascorbate may
significantly change. For instance, in tobacco plants infected with TMV, ascorbic acid
content decreased in inoculated leaves, whereas its oxidized form significantly increased, a
result attributed to virus-induced oxidative stress (Fodor et al., 1997).
Both ascorbate and GSH can be readily oxidized by ROS to, respectively,
monodehydroascorbate (MDA) and dehydroascorbate (DHA), and oxidized glutathione
(GSSG – glutathione disulphide), a means of alleviating oxidative damage. Through the
activity of specific enzymes of the ascorbate-glutathione cycle, GSH and ascorbate can be
regenerated. The reduction of GSSG and MDA is accomplished by glutathione reductase
(GR) and monodehydroascorbate reductase (MDAR) respectively, using NADPH as
reductant. DHA, however, is reduced by the enzyme dehydroascorbate reductase (DHAR)
that uses GSH as substrate. It is crucial for the plant cell to maintain high levels of reduced
ascorbate and GSH for effective ROS scavenging (Apel and Hirt, 2004) (Fig. 3).
1.2.3
ROS-scavenging enzymes
Among the enzymes acting as ROS scavengers, the following have been well documented;
superoxide dismutase (SOD, EC 1.15.1.1), ascorbate peroxidase (APX, EC 1.11.1.11),
glutathione peroxidase (GPX, EC 1.11.1.9), and catalase (CAT, EC 1.11.1.6). Under
conditions of stress, such as infection by pathogenic agents, activity or/and level of
antioxidant enzymes can be modulated.
Regarded as among the most powerful enzymatic antioxidants in cells, SODs are
widespread in all aerobic organisms in which oxidative stress can occur. It is the first line
of defence against stress-induced ROS, catalysing the dismutation of O2•- to H2O2 (Fig. 3).
SODs are often up-regulated under abiotic and biotic stress, playing critical roles in plants’
survival (Apel and Hirt, 2004; Gill and Tuteja, 2010), although this is not always the case.
SOD activity strongly declined in Phaseolus vulgaris after infection with an non-necrotic
isolate of White clover mosaic virus (WClMV) (Clarke et al., 2002), whereas Radwan et al.
(2010) showed increased activity in Bean yellow mosaic virus (BYMV)-infected Vicia faba
11
leaves. In cucumber leaves infected with CMV, chloroplastic and mitochondrial SOD
activity rose up to 85%. Similar results were observed in CMV-infected tomato leaves
(Song et al., 2009). Furthermore, in Nicotiana benthamiana plants infected with a virulent
strain of PMMoV, the activity of different SOD isoforms was diminished as compared to
control plants. However, when infection was induced by a less virulent strain of PMMoV,
SOD activity dramatically increased (Hakmaoui et al., 2012). In addition, a susceptible
apricot cultivar infected with Plum pox virus (PPV) showed decreased levels of SOD
activity in the apoplastic space, in contrast to a resistant cultivar that revealed a significant
rise in the enzyme activity (Díaz-Vivancos et al., 2006).
H2O2, accumulating as SOD removes superoxide anions, is subsequently reduced by APX,
GPX and CAT. CAT is found in almost all aerobic organisms. In plants, catalases are
present in various enzymatic forms (or isozymes), and are responsible for the conversion of
H2O2 to H2O (Fig. 3), a reaction that does not require any reducing equivalents (Apel and
Hirt, 2004; Mhamdi et al., 2010; Scandalios et al., 1997). CAT activity is often seen to be
altered when plants are attacked by pathogens. It declined in P. vulgaris in response to
WClMV infection (Clarke et al., 2002) whereas it was significantly induced in BYMVinfected V. faba leaves (Radwan et al., 2010).
APX has been reported in numerous higher plants, several isoforms having been identified
(Shigeoka et al., 2002). Its role is to detoxify H2O2, for which it has a high affinity (Gill and
Tuteja, 2010). APX reduces H2O2 utilizing ascorbate as specific electron donor, a reaction
resulting in water and MDA generation (Fig. 3) (Apel and Hirt, 2004; Shigeoka et al.,
2002). Its expression has been shown to increase under different stress conditions (Gill and
Tuteja, 2010), a factor that may be crucial in plant-pathogen interaction. As evidenced by
Conklin et al. (2001), levels of several APX mRNAs were shown to peak as a consequence
of elevated ROS concentration. Additionally, in Cucumis sativus and Cucurbita pepo
infected with both CMV and ZYMV marked increases in APX activity were observed
(Smirnoff and Wheeler, 2000).
12
GPX is found in nearly all subcellular compartments (cytosol, chloroplasts, mitochondria)
and acts in a similar fashion to APX by catalysing the reduction of H2O2. It is also capable
of detoxifying products derived from lipid peroxidation (Gill and Tuteja, 2010). Using
GSH as electron donor, GPX mediates the conversion of H2O2 to water, GSH being
oxidized to GSSG (Fig. 3). GSH is subsequently regenerated from GSSG via the activity of
glutathione reductase, thus completing the GPX cycle (Apel and Hirt, 2004). Studies have
reported shifts in GPX activity in response to environmental stress. In barley root tips, root
growth inhibition treatments using heavy metals strongly induced GPX activity (Halušková
et al., 2009). Transgenic tomato plants over-expressing an animal GPX were less affected
by mechanical wounding than control plants (Herbette et al., 2011). Similarly, tobacco
plants engineered to over-express a tomato phospholipid hydroperoxide GPX showed high
resistance to a fungal necrotroph as well as against salt stress (Chen et al., 2004). For
efficient enzymatic activities of both APX and GPX in ROS-scavenging process, the
regeneration of ascorbate and GSH in the ascorbate-glutathione cycle is required.
In plants, glutathione reductase (GR, EC 1.8.1.7) is mainly found in chloroplasts. It is
importantly involved in the ascorbate-glutathione cycle as it catalyzes the reduction of
GSSG to GSH, using NADPH as reductant (Fig. 3). Thus, GR indirectly participates in
defence against oxidative stress by regenerating GSH (Ding et al., 2009; Gill and Tuteja,
2010). GR activity has been reported to be influenced by various environmental conditions.
Edwards et al. (1994) showed increased levels of GR proteins in pea leaves treated with
ozone. In PMMoV-infected Nicotiana benthamiana, GR activities significantly dropped as
compared to controls, but eventually reached normal levels at an advanced stage of the
infection (Hakmaoui et al., 2012).
Glutathione S-transferases (GSTs, EC 2.5.1.18) are a large family of enzymes that catalyse
the conjugation of GSH to electrophilic xenobiotics that are often toxic for plant cells
(Dixon et al., 2010; Marrs, 1996). Some GSTs are suggested to act as antioxidants whose
expression/activity might be significantly enhanced by H2O2 (Noctor et al., 2012). Besides
their implications in pathogen-related stress responses and oxidative stress, GSTs play
pivotal roles in normal plant metabolism (Marrs, 1996).
13
1.2.4
Protein carbonylation & lipid peroxidation as markers of
oxidative stress
Proteins are preferential targets of ROS, representing up to 70% of the cellular components
being oxidized (Rinalducci et al., 2008). Oxidation of proteins is regarded as a marker of
oxidative damage, of which protein carbonylation is a frequent protein modification process
(Dalle-Donne et al., 2003; Moller et al., 2007). ROS-induced protein carbonylation impairs
structure and/or function of targeted proteins. It involves the oxidation of certain amino
acids side-chains such as lysine, proline, arginine and threonine, to which aldehyde and
ketone groups are formed (Bahramikia et al., 2009; Juszczuk et al., 2008; Lounifi et al.,
2012; Qiu et al., 2008; Rinalducci et al., 2008). Furthermore, sulphur-containing amino
acids, i.e. cysteine and methionine, are known to be amongst the most oxidative-susceptible
residues (Rinalducci et al., 2008). Protein carbonylation may play important roles in diverse
biological processes, with carbonylated protein levels often increasing in plants stressed by
different abiotic and biotic factors (Lounifi et al., 2012). As evidenced by Qiu et al. (2008),
elevated CO2 concentrations induced increased leaf protein carbonylation in A. thaliana and
soybean. In N. benthamiana infected with a virulent strain of PMMoV, carbonylated
protein content was also drastically increased (Hakmaoui et al., 2012). Likewise, in a PPVinfected susceptible apricot cultivar, the level of oxidized proteins was greatly increased,
which correlated with decreased antioxidant enzyme levels (Hernández et al., 2006).
Although oxidative modification of proteins may be detrimental for plant cells, it has been
pointed out that it might also be a tightly regulated and reversible process involved in
normal cell homeostasis. For protein carbonylation though, no evidence for reversibility has
been found (Rinalducci et al., 2008).
One major effect of ROS on plant cells is the peroxidation of membrane lipids, a process in
which highly toxic lipid peroxides are formed (Marrs, 1996). The production of superoxide
anions largely contributes to lipid peroxidation (Rustérucci et al., 1996). Polyunsaturated
fatty acids (PUFAs) found in membrane lipids are particularly susceptible to such
peroxidation. The products of oxidative stress-induced lipid peroxidation enhance free
radical reactions that can greatly affect the integrity of phospholipid membranes by
impairing their physicochemical properties (Catalá, 2009). Rustérucci et al. (1996) showed
14
increased lipid peroxidation with accumulation of lipid peroxides in tobacco plants treated
with the necrosis-inducing elicitin cryptogein. Interestingly, the double infection of N.
benthamiana with Potato virus X (PVX) and Potato virus Y (PVY) induced high levels of
lipid peroxidation, whereas single infections did not show any significant changes (GarcíaMarcos et al., 2009). Likewise, PPV-infected pea plants had augmented lipid peroxidation
15 days post-inoculation (Díaz-Vivancos et al., 2008). The process of lipid peroxidation, in
conjunction with ROS formation, may be involved in cell death mechanisms as it occurs
during aging or senescence of plant tissues (Rustérucci et al., 1996). Furthermore, radical
products formed by lipid peroxidation can induce the oxidation of pigment molecules that
results in the development of yellowing or mosaic patterns on infected leaves (RiedleBauer, 2000).
In incompatible plant-virus interactions, the formation of ROS, often related to the HR with
localized programmed cell death of infected cells, plays a central role in virus resistance.
However, in compatible interactions, the involvement of ROS and antioxidants during the
infection process and resistance mechanisms has yet to be understood, with contradictory
observations showing enhanced or decreased activity of antioxidant enzymes (Hernández et
al., 2006).
15
GSH
Ascorbate
ROS
ROS
ROS
H2O2
GSSG
GR
NADPH
MDA
MDAR
NADPH
DHA
DHAR
O2•-
Ascorbate
H2O + ½ O2
H2O2
GSH
H2O2 + Ascorbate
GSH
SOD
CAT
APX
H2O + MDA
Ascorbate
H2O2 + GSH
GPX
H2O + GSSG
Figure 3 Interactions between ROS and scavenging system. APX = ascorbate peroxidase, CAT = catalase, DHA = dehydroascorbate, DHAR = dehydroascorbate reductase,
GPX = glutathione peroxidase, GR = glutathione reductase, GSH = glutathione, GSSG = glutathione disulphide, H2O2 = water, H2O2 = hydrogen peroxide, MDA =
monodehydroascorbate, MDAR = monodehydroascorbate reductase, NADPH = nicotinamide adenine dinucleotide phosphate, O2 = oxygen, O2•- = superoxide, ROS =
reactive oxygen species, SOD = superoxide dismutase (Apel and Hirt, 2004; Gill and Tuteja, 2010).
16
1.3 Project introduction
For all experiments, the legume plant white clover (Trifolium repens L.) and the plant virus
White clover mosaic virus (WClMV) were used. Two cultivars of white clover, respectively
‘Huia’ and ‘Kopu II’, were employed.
1.3.1
White clover (Trifolium repens L.)
Also referred as Dutch clover, white trefoil, creeping trifolium, ladino clover, or even
honeysuckle clover, Trifolium repens L. is an herbaceous prostrate perennial plant,
although it might behave as an annual plant under environmental stress conditions. It is a
natural tetraploid (4n = 32 chromosomes) that belongs to the Fabaceae family, and it is
regarded as one of the most agronomically important clover species. Initially thought to
have been domesticated 400 years ago in the Netherlands, white clover was spread around
the world by European immigrants. Nowadays, its range is mainly found in Western
Europe, North America, Australia and New Zealand (Office of the Gene Technology
Regulator, 2008). As a nitrogen-fixing legume, white clover is an important component of
agriculture in temperate ecosystems (Andrews et al., 2007; Mouradov et al., 2007) and it is
broadly used as cover crop and organic fertilizer (Carlsen and Fomsgaard, 2008).
The structural unit of white clover is called the stolon, defined as a creeping stem that
grows on the soil surface and consists of a series of internodes. The proliferation of
stoloniferous stems allows for vegetative propagation. Each node is characterized by a
trifoliate leaf, two root primordia that may develop adventitious roots when soil moisture
conditions are favourable, and an axillary bud capable of growing into a lateral stolon
during active vegetative growth (Fig. 4). The leaflets that compose a clover leaf are usually
elliptical or egg/heart-shaped, with subtle serrate margins. Leaflet size varies from less than
1 cm to more than 2 cm long depending on type and cultivar as well as environmental
conditions. Some clover leaves are uniformly green, while others display whitish coloration
drawn on the upper surface in a V-shaped pattern (Fig. 5A). Emerging for active apical
buds, flowers are borne by a long peduncle and organised in globular racemes. Each
inflorescence is made of about 20-40 white florets (Fig. 5B) (Office of the Gene
Technology Regulator, 2008).
17
Inflorescence
Trifoliate leaf
Peduncle
Emerging
inflorescence
Internode
Node
Adventitious
root
2 cm
0.5 cm
Figure 4 Stolon characteristics of white clover (M. Corral).
The ability of white clover to fix atmospheric nitrogen (N2) is conferred by the soil bacteria
Rhizobium sp. These symbiotic bacteria are contained in specific structures called root
nodules (Fig. 6). The enzyme involved in N2 fixation is the nitrogenase, the activity of
which is inactivated under elevated O2 concentration. However, aerobic respiration is
required to supply sufficient amounts of ATP to the enzyme. To counteract this problem
and regulate the flux of O2 to the bacteria, legume infected-cells have evolved barriers to
limit oxygen inhibition and enhance respiration simultaneously (Hunt and Layzell, 1993;
Pugh et al., 1995; Ronson and Lowther, 1996).
As reviewed by Ledgard et al. (2009), white clover grown in temperate pastoral systems
may fix about 10 to 300 kg of N2 per hectare per year. Nitrogen can be released through
root exudates, from senescent tissues, as well as from grazing animal excrements, and thus
be available for other pasture plants (Andrews et al., 2007). Accordingly, white clover
substantially increases pastures’ nutritional value through nitrogen inputs (Denny and Guy,
18
2009). Interestingly, when comparing a pasture growing ryegrass in association with white
clover and a pasture growing ryegrass only but supplied with 200 kg N.ha-1.annum-1,
Andrews et al. (2007) reported that plant dry matter, and milk production/quality from
cattle are likely to be similar in both systems. Consequently, pastures with grass and clover
grown in conjunction show lesser needs in regards to N-fertiliser inputs. Greenhouse gas
emissions are therefore reduced as N-fertiliser production uses fossil fuels to drive high
temperature and high pressure chemical reactions, whereas N2 fixation by legumes only
uses photosynthetically produced carbon at ambient temperature and normal atmospheric
pressure (Ledgard et al., 2009).
A
V-shaped mark
Serrate margin
2 cm
B
Florets
2 cm
Figure 5 Pictures showing leaves (A) and inflorescences (B) of white clover (M. Corral).
19
0.5 cm
4 cm
Figure 6 Root from white clover showing nodules (M. Corral).
White clover is also important in terms of seed and honey production (Denny and Guy,
2009), it is a valuable source of proteins and minerals for livestock, and it markedly
improves yields of dairy, meat and wool products (Office of the Gene Technology
Regulator, 2008). Harris et al. (1997) reported that white clover’s nutritive value was
greater than grasses as it has low levels of structural carbohydrate and high content in
digestible protein, enhancing protein and metabolisable energy intakes in cattle along with
improved digestibility and increased milk production. Pastoral industries are of prime
importance for New Zealand’s economy, providing animal products of exceptional quality
being highly competitive on international markets. As a key component of pastures, white
clover plays a vital role in such competitiveness. Indeed, based on all benefits mentioned
above, annual financial contribution of T. repens of up to $NZ 3 billion have been
estimated in New Zealand (Caradus et al., 1996).
20
Clovers are susceptible to a number of plant viruses. In New Zealand, up to 12 viruses
infecting white clover have been reported, including: White clover mosaic virus (WClMV),
Alfalfa mosaic virus (AMV), Lucerne Australian latent virus (LALV), Clover yellow vein
virus (CYVV), Zucchini yellow mosaic virus (ZYMV), Watermelon mosaic virus (WMV),
Red clover necrotic mosaic virus (RCNMV), Soybean dwarf virus (SbDV), Beet western
yellows virus (BWYV), Tomato spotted wilt virus (TSWV) and White clover virus L
(Denny and Guy, 2009). More recently, Red clover vein mosaic virus (RCVMV) has also
been detected in T. repens (J. Fletcher, 2012; personal communication).
1.3.2
White clover mosaic virus: a foe for clover species
Belonging to the family Alphaflexiviridae and the genus Potexvirus (Forster et al., 1988),
White clover mosaic virus (WClMV) is considered an important pathogenic agent infecting
clovers worldwide (Potter, 1993). It was first reported in white clover in 1935 by Pierce
WH and has been shown to have no insect vectors. Instead it spreads from host to host
through mechanical transmission, mostly via animal grazing and trampling, and agricultural
practices (Van Molken et al., 2012). Other plant viruses have been reported to be
transmitted by contact, including TMV, PPMoV, PVX, and Rice yellow mottle virus
(RYMV), among others (Sacristán et al., 2011; Sarra et al., 2004).
The virion is made of a flexuous filamentous rod of about 480 nm long (Beck et al., 1990)
(Fig. 7) enclosing a positive-sense ssRNA (of about 2.106 Dalton) that is capped and
polyadenylated to prevent RNA degradation (Clarke et al., 2000). Its complete genome has
been sequenced and consists of 5843-46 nucleotides (depending on the WClMV strain,
three isolates having been sequenced so far) (Nakabayashi et al., 2002), including 5 distinct
open reading frames (ORFs) (Forster et al., 1988) (Fig. 8). The capsid of potexviruses is
formed of about 1000-1500 protein subunits, with a molecular weight of 18-27 kDa (King
et al., 2011).
21
500 nm
Figure 7 Observation of rod-shaped virus particles of White clover mosaic virus with a transmission electron
microscope at 38,000x magnification (M. Corral & R. Easingwood, Otago Centre for Electron Microscopy).
In the Potexvirus group, cell-to-cell movement requires the activity of 3 overlapping ORFs
referred as to the triple gene block (TGB). Although the presence of all 3 TGB proteins is
crucial for cell-to-cell movement, only the so-called TGB protein 1 (TGBp1) is able to
cross over the plasmodesmata along with the coat protein. Indeed, TGBp1 is capable of
binding to viral RNAs and increasing the size exclusion limit (SEL) of plasmodesmata,
therefore enhancing viral transport. Likewise, the Potato virus X (PVX) TGBp1 was shown
to interfere with plant defence mechanisms by suppressing host-driven silencing responses
(Lough et al., 2001).
5’
RNA-dependent RNA polymerase
ORF 3
ORF 2
ORF 4
CP
AAA3’
Triple gene block
(TGB)
Figure 8 Organisation of WClMV genomic RNA. ORF 1 encodes for an RNA- dependent RNA polymerase
that has homology with the one of TMV. The proteins involved in cell-to-cell movement are encoded by the
triple gene block (TGB) that clusters ORF 2, ORF 3 and ORF 4. The CP is translated from a subgenomic
RNA that is located within ORF 5 (Clarke et al., 2000).
22
In white clover, WClMV adversely impacts both growth and developmental processes. The
virus induces a reduction in stolon branching, root and leaf biomass, although it does not
seem to affect the development of flowers (Van Molken and Stuefer, 2011). Typical
symptoms resulting from the infection include the development of light/dark-green mosaic
patterns on the upper surface of infected leaves (Fig. 9). Stolons are known to store
carbohydrates. Hindering the production of such reserve organs would therefore affect the
plant’s growth and competitive ability. For this reason, stolon branching is seen as a
decisive factor that determines growth and yield of white clover (Potter, 1993). Nodulation
and N2-fixation capacity of white clover may also be dramatically impeded by WClMV
infection (Dudas et al., 1998; Guy et al., 1980). It therefore appears clear that white clover
resistance to WClMV must be investigated. Although our knowledge about such resistance
is scarce, some already published work has provided useful and promising information. As
suggested by Scott (1982), one possibility would be the breeding, testing and selection of
infected plants with low virus concentration, a trait that could be related to a certain level of
viral resistance (Scott, 1982). Likewise, as acknowledged by the same author, Barnett and
Gibson (1975) suggested the hybridisation between WClMV-resistant Trifolium ambiguum
Bieb (Kura clover) and T. repens to introduce the resistance to the virus. Molecular
engineering may as well be used to obtain resistant transgenic lines; examples include the
expression of mutated viral genes (e.g. triple gene block) (Beck et al., 1994), several
constructions of the coat protein gene (Dudas et al., 1993) or the entire replicase gene to
confer resistance to WClMV and other viruses (Guy and Forster, 1996).
1.4 Thesis objectives
The aims of the present study are focused on how White clover mosaic virus affects the
fitness and the nutritional value of white clover (Trifolium repens L.). The first experiment,
of which Chapter 3 relates, is based on the comparison of the effect that WClMV has on
two cultivars of T. repens. We aimed to look at; (1) whether the virus affects growth,
physiology and biochemistry of white clover, in particular how the host plant copes with
oxidative stress; and (2) whether these effects differ between the two cultivars. A second
experiment was conducted to give insights into the extent to which a root-flooding
treatment, as an abiotic stress, may influence white clover oxidative stress and WClMV
23
infection, as detailed in Chapter 4. In this respect, we investigated (1) whether waterlogging
stress dramatically affects the plant, and (2) whether the response between infected and
uninfected plants differs.
Figure 9 Leaf symptoms induced by WClMV (leaf from white clover Huia at 43dpi) (M. Corral).
Responses towards WClMV greatly depend on white clover genotypes, genotypic variation
being an important trait in plant responses to viral infection. It has been emphasized that
viruses exert a strong selection pressure in natural plant populations, thus enhancing and/or
maintaining genotypic diversity (Van Molken and Stuefer, 2011).
24
2. Materials and Methods
2.1 Study organisms
For all experiments, the legume plant white clover (Trifolium repens L.) and the plant virus
White clover mosaic virus (WClMV) were used. Commercially obtained seeds of white
clover cv. Huia and cv. Kopu II were sown in a glasshouse. Four weeks post-sowing, 51
seedlings from Huia and 51 seedlings from Kopu II were randomly selected and
transplanted into 1.5 L pots filled with a mix of potting compost and coarse sand (50/50).
Pots were arranged in two blocks (benches), their position having been randomized once a
week (refer to sections 3.2 and 4.2 in Chapter 3 & 4, respectively). The plants were watered
everyday and grown in a 16-h light/8-h dark regime (under mixed lighting, Phillips SON-T
Agro) at approximately 17 °C and 80% relative humidity. WClMV inoculum was originally
obtained from a naturally field-infected white clover collected in Waitati, a site located on
the South Island of New Zealand. As WClMV does not produce local lesions, but instead
infects the plant as a whole, single lesion isolates could not be obtained. This plant and a
subset of the Huia and Kopu II plants were tested, from time to time, for the presence of
other clover viruses (ELISA results were negative, data not shown).
2.1.1
WClMV inoculation
Twenty-six replicates from each cultivar were randomly chosen and mechanically infected
with the virus 3 weeks post-transplantation, for which at least 5-8 trifoliate leaves per plant
had formed. WClMV-infected plant material was ground using a mortar and a pestle in
inoculation buffer consisting of 10 mM sodium phosphate and 10 mM sodium sulphite.
Leaves were dusted with carborundum, used as abrasive agent, and subsequently rubbed
with the infected cell sap. Control plants, a total of twenty-five replicates per cultivar, were
mock-inoculated with buffer solution only. In order to detect the presence of WClMV, all
inoculated plants as well as randomly selected controls were tested by Double Antibody
Sandwich – Enzyme Linked ImmunoSorbent Assay (DAS-ELISA).
25
2.1.2
WClMV detection: DAS-ELISA protocol
Plants inoculated with WClMV were tested by DAS-ELISA according to the method of
Clark and Adams (1977). Prior to the analysis, multi-well plates were coated with 100 µl of
WClMV-Immunoglobulin G (IgG) (1/2000) diluted in coating buffer (1.59 g-1.l-1 Na2CO3,
2.93 g-1.l-1 NaHCO3, 0.2 g-1.l-1 NaN3, pH 9.6). From each sample tested, one or two leaves
were collected and ground into grinder bags with PBS-etc extraction buffer (containing 100
ml PBS 10x, 900 ml RO grade water, 1 ml Tween 20, 20 g polyvinylpyrrolidone PVP-40,
and 2 g bovine serum albumin). Phosphate-buffered saline 10 times concentrated (PBS
10x) was made of 80 g NaCl, 2 g KH2PO4, 29 g Na2HPO4.12H2O, 2 g KCl, and 2 g NaN3,
in 1 litre (pH 7.4). The plates were washed several times using PBS-T washing buffer (100
ml PBS 10x, 900 ml RO grade water, and 1 ml Tween 20), and 100 µl of sap prepared in
PBS-etc was subsequently loaded in each well and incubated for 2 hours at room
temperature. 5 µl of conjugated WClMV antibodies (from antibodies raised to an Auckland
isolate of WClMV by Associate Professor Paul Guy, (Denny and Guy, 2009)) were
incubated in 11 ml of healthy sap (1/20 in PBS-etc) for 1 hour at 37 °C, in order to allow
the unspecific binding sites of the antibodies to be occupied. The sample was then
centrifuged 20 minutes at 5,000 rpm, and 10 ml of supernatant was mixed with 0.1 g of
skim milk. After successive post-incubation PBS-T washes of the sap-loaded plates, 100 µl
of conjugated antibodies were added into each well, and the plates were allowed to incubate
for 2 hours at room temperature. Once again, the plates were washed consecutively, using
water first and then PBS-T buffer, and 100 µl of substrate buffer (containing 97 ml
diethanolamine, 800 ml RO grade water, adjusted to pH 9.8 using HCl), in which
phosphatase substrate PNP (p-nitrophenyl phosphate) tablets were dissolved, were added
into each well. Finally, the plates were incubated for 30 minutes at room temperature for
colour development.
2.1.3
Observation of WClMV particles
Leaf tissue collected from mechanically WClMV-infected white clover was ground in
distilled water at approximately 1/20 (w/v). 20 µl drops were placed on carbon coated
electron microscopy grids for 2 minutes and then blotted. 20 µl drops of 2% sodium
phosphotungstate (pH 6.5) were placed on the grids for 2 minutes, the latter being
26
subsequently blotted, dried and examined in a Philips Transmission Electron Microscope at
38,000x magnification.
2.1.4
Measurement of photosynthetic efficiency and growth
For both uninfected and infected plants (randomly selected), maximum photochemical
efficiency (Fv/Fm) was measured by averaging readings from 3 leaves per plant, using a
PAM-2000 portable chlorophyll fluorometer (manufactured by Heinz Walz GmbH,
Germany). Before any measurement, the plants were dark-adapted for 30 minutes in which
the photosynthetically active radiation (PAR) was below 15 µmol.s-1.m-2. Fv/Fm values were
determined and averaged for 10 leaves of an uninfected white clover Kopu II plant,
providing an “unstressed-control” Fv/Fm reference value of 0.770.
At each harvest (for which further details are given in Chapter 3 & 4 for Experiment 1 and
Experiment 2, respectively), above-ground fresh weight was measured and samples (i.e.
above-ground plant material) from each plant were taken for biochemical assays and
nutritional analyses. For the biochemical assays, samples were frozen in liquid nitrogen and
then stored at -80 °C before analysis. For the nutritional analyses, performed only in
Experiment 1, samples were placed in a drying oven for 3 days at 50 °C, reweighed and
stored in dry environment prior to analysis. From the dried plant materials, relative dry
matter content (%) was calculated to determine the absolute dry matter content of each
plant (g per plant) (see below). In Experiment 2 only, root samples were also collected for
biochemical assays. Dry matter content was calculated as followed:
Dry weight of the sample
Relative DMC (%) =
Fresh weight of the sample
Absolute DMC (g.plant -1) = (Relative DMC) x (Total fresh weight of the plant)
27
2.2 Biochemical assays
Frozen plant materials were ground in liquid nitrogen using a mortar and a pestle until a
fine powder was obtained. Enzymatic and non-enzymatic antioxidants, as well as total
protein content, protein carbonyls and lipid peroxides were then extracted and analyzed
according to the following protocols. For all the centrifugation steps described below, an
Eppendorf 5417R centrifuge fitted with a F45-30-11 rotor was used.
2.2.1
Enzymatic antioxidants extraction and analysis
CAT, SOD, GST, GPX, and GR were extracted by homogenising 100 mg of powdered
tissue in 900 µl of ice-cold extraction buffer (100 mM potassium phosphate (pH 7.5), 0.25
mM K2EDTA, 1 mM PMSF, 0.05% (v/v) Triton X-100 and 2% (w/v) Polyclar AT
(SERVA Chemicals Ltd)). For APX extraction, 5 mM of ascorbate was added to the buffer.
Homogenates were mixed using a vortex mixer and centrifuged at 14,000 rpm for 15
minutes at 4 °C, the resulting supernatants being subsequently divided into 120 µl aliquots
and stored at -80 °C before analysis.
CAT activity was determined using the chemiluminescent method of Maral et al. (1977), as
adapted by Janssens et al. (2000) for measurements in microplates, with minor
modifications (Schweikert and Burritt, 2012). 50 µl of extract, diluted extract or purified
bovine liver CAT (Sigma-Aldrich, St. Louis, MO, USA) was mixed with 100 µl of 100 mM
phosphate buffer (pH 7.0, containing 100 mM NaEDTA and 10-6 M H2O2), followed by an
incubation step at 25 °C for 30 minutes. Then, 50 µl of a solution containing 20 mM
luminal and 11.6 units.ml-1 of horseradish peroxidase (Sigma-Aldrich, St. Louis, MO,
USA) was added into each well and light emission, for which the intensity was proportional
to the amount of H2O2 remaining, was measured. A regression analysis was used to prepare
a standard line correlating standard CAT activities to the intensity of light emission. CAT
activities were calculated with reference to the standard line and expressed as µmol of H2O2
consumed.mg-1 protein.
SOD was assayed via the method of Banowetz et al. (2004) with minor modifications
(Schweikert and Burritt, 2012). 50 µl of extract, diluted extract or standard (prepared from
28
bovine liver SOD, Sigma-Aldrich, St. Louis, MO, USA) was mixed with 125 µl of prepared
reaction solution containing piperazine-1,4-bis(2-ethanesulfonic acid) (Pipes) buffer, 0.4
mM o-dianisidine, 0.5 mM diethylenetriaminepentaacetic acid (DTPA), and 26 µM
riboflavin (pH 7.8). One unit of SOD corresponded to the amount of enzyme that inhibited
the reduction of cytochrome c by 50% in a coupled system with xanthine oxidase at pH 7.8
and 25 °C. The absorbance at 450 nm (A450) was measured immediately (t = 0 min), the
samples were illuminated with an 18 W fluorescent lamp placed 12 cm above the plate for
30 minutes (t = 30 min) and the A450 was measured again. A regression analysis was used
to prepare a standard line relating SOD activity to the change in A 450 and SOD activities of
the extracts were calculated with reference to the standard line and expressed as units of
SOD.mg-1 protein.
GST activity was determined according to the photometric 1-chloro-2,4-dinitrobenzene
(CDNB) method of Habig et al. (1974) with minor modifications (Schweikert and Burritt,
2012). The absorbance at 340 nm (A340) was measured every 30 seconds for 3 min, the
plate being shaken automatically before each reading. The change in A340 per minute was
calculated and converted into nmoles of CDNB conjugated to GSH.min-1.mg-1 protein
using the extinction coefficient of the resulting S-2,4-dinitrophenylglutathione (DNPG):
E340 9.6 mM-1.cm-1.
GPX activity was assayed using the spectrophotometric method of Paglia and Valentine
(1967) with modification for use with a microplate reader (Phang et al., 2011). 50 µl of
extract, diluted extract or standard (GPX from bovine erythrocytes, Sigma-Aldrich, St.
Louis, MO, USA, in extraction buffer) was mixed with 170 µl of assay buffer containing 50
mM Tris-HCl buffer (pH 7.6), 5 mM EDTA, 0.14 mM NADPH, 1 mM GSH and 3 units/ml
glutathione reductase (from wheat germ, Sigma-Aldrich, St. Louis, MO, USA). The
reaction was initiated by the addition of 20 µl t-butyl hydroperoxide to give a final
concentration of 0.2 mM. The consumption of NADPH was monitored at 340 nm (A340)
every 30 seconds for 3 minutes, with the plate shaken automatically before each reading.
GPX activities in the extracts were calculated with reference to a standard line constructed
with GPX purified from bovine erythrocytes, and expressed as nmol.mg-1 total protein.
29
GR activity was determined according to the method of Cribb et al. (1989) with minor
modifications (Schweikert and Burritt, 2012). 50 µl of extract, diluted extract or standard
(wheat germ GR, Sigma-Aldrich, St. Louis, MO, USA, in homogenization buffer) was
mixed with 150 µl of 100 mM sodium phosphate buffer (pH 7.6, 0.1 mM 5,5’-dithiobis(2nitrobenzoic acid) (DTNB), 10 µl NADPH (10 mg/ml, 12 mM)). 10 µl of GSSG (1 mg/ml,
3.25 mM) was injected to initiate the reaction, and the absorbance at 415 nm (A415) was
measured every 30 seconds for 3 min, the plate being automatically shaken before each
reading. The rate of increase in A415 per minute was calculated and regression analysis used
to prepare a standard line relating standard GR activities to the absorbance. GR activities
were calculated by referring to the standard line and expressed as nmol of GSSG
reduced.min-1.mg-1 total protein.
APX activity was assayed, according to Rao et al. (1996) with minor modifications
(Schweikert and Burritt, 2012), by following the decrease in the absorbance at 290 nm
(A290) as ascorbate disappeared. 50 µl of extract was mixed with 200 µl of 100 mM
potassium phosphate buffer (pH 7.0) containing 0.5 mM ascorbate and 0.2 mM H2O2. APX
activity (expressed as µmol.min-1) was calculated using an extinction coefficient of 2.8
mM-1.cm-1, corrected for the calculated path-length of the solution (0.6 cm).
2.2.2
Non-enzymatic antioxidants extraction and analysis
2.2.2.1 Extraction and analysis of ascorbate
Ascorbate was extracted by homogenising 100 mg of ground tissue in 1 ml of ice-cold 5%
(w/v) metaphosphoric acid. The samples were mixed using a vortex mixer and spun at
14,000 rpm for 15 minutes at 4 °C. The obtained supernatant was then divided into 200 µl
aliquots and stored at -80 °C prior to analysis. Reduced and total ascorbate levels were
assayed using the microplate-adapted method described by Gillespie and Ainsworth (2007).
2.2.2.2 Extraction and analysis of glutathione
Glutathione content was extracted by homogenising 100 mg in 1 ml of ice-cold 5% (w/v)
sulfosalicylic acid. Homogenates were thoroughly mixed and centrifuged at 14,000 rpm for
15 minutes at 4 °C. The resulting supernatant was divided into 200 µl aliquots and stored at
30
-80 °C before analysis. Reduced and total glutathione levels were determined using the
enzymatic recycling method adapted for microtitre plates described by Rahman et al.
(2006).
2.2.3
Protein content and protein carbonyls extraction and analysis
Total protein content and protein carbonyls were extracted by homogenising 100 mg of
powdered tissue in 900 µl of ice-cold extraction buffer containing 100 mM potassium
phosphate, 0.1 mM Na2EDTA, 1 mM PMSF, 0.3% TritonX-100 and 1% PVPP. The extract
was then mixed and centrifuged at 14,000 rpm for 15 minutes at 4 °C. The supernatant
obtained was dispensed into 120 µl aliquots and stored at -80 °C before analysis. Protein
carbonyl contents were analysed according to the 2,4-dinitrophenylhydrazine method of
Reznick and Packer (1994), adapted for measurement in a microplate reader. Levels of
protein carbonyls were determined using the extinction coefficient of DNPH at 370 nm
(0.022 µmol-1.cm-1), corrected for the calculated path-length of the solution (0.6 cm). The
protein content of the extracts was determined using a Lowry protein assay (Fryer et al.,
1986). Protein carbonyl contents were expressed as nmol carbonyls.mg-1 protein.
2.2.4
Lipid peroxides extraction and analysis
Lipid peroxides were extracted by homogenising 100 mg of powdered tissue in 600 µl of
methanol:chloroform (2:1, v/v). The tissue was left to stand for 1 minute, and 400 µl of
chloroform was then added and mixed for 30 seconds. 400 µl of deionised water was added
to the extract and mixed again for 30 seconds. Phase separation was obtained by
centrifuging the homogenate at 13,000 g for 30 seconds. The lower chloroform phase, in
which lipid peroxides are contained, was transferred into a new tube and stored at -80 °C
prior to analysis. For each sample, 50 µl of aliquot was transferred to a glass microtitre
plate well and lipid peroxide contents were determined using the ferric thiocyanate method
(Mihaljevic et al., 1996), adapted for measurement in a microplate reader, with absorbance
measured at 500 nm. Lipid peroxide contents were expressed as nmol of lipid peroxide.g-1
FW.
31
2.3 Nutritional analysis
Dried samples from each cultivar and treatment (i.e. uninfected vs WClMV-infected)
(Experiment 1 only) were pulled together (3 samples in 1) as too small amounts of plant
material were obtained after the drying step. The “meta-samples” were subsequently finely
ground, stored in vials and sent to R J Hill Laboratories Limited (Hamilton, New Zealand)
for pasture feed quality analysis, using Near-Infrared (NIR) Spectroscopy methods (as
listed in Table 1, provided by R J Hill Laboratories Ltd).
2.4 Root cross-sections
Root samples were collected from non-waterlogged and waterlogged plants 5 weeks postwaterlogging treatment. Fine cross-sections of roots were obtained using razor blades and
stained with toluidine blue. The pictures were taken using a Zeiss microscope equipped
with a digital camera.
2.5 Statistical analysis
Statistical analyses were performed using SPSS Statistics 20 for Windows. All data were
analysed using two-way analysis of variance (ANOVA) and tested for normality
assumption (Kolmogorov-Smirnov’s test) and homogeneity of variance (Levene’s test).
Minor violations of parametric assumptions were observed in a few cases and were judged
insignificant. Data from growth parameters (Experiment 1 & 2) and biochemical assays
(Experiment 1 only) were analysed using independent samples t-tests with the level of
significance P < 0.05 (*) to detect differences between controls and virus-treated samples.
For multiple-level interactions, i.e. data from nutritional analyses (Experiment 1) and
biochemical assays (Experiment 2), separate generalized linear models (GLMs) followed
by Bonferroni post-hoc testing were used, with different letters in graphs and tables
indicating significant differences at P < 0.05. All values are presented as means ± standard
error of the mean (SE), except for nutritional data for which S.D. are displayed in tables.
32
Table 1 Summary of methods for Pasture Feed Quality Analysis (provided by R J Hill Laboratories Ltd).
Sample Type: Plant
Test
Method Description
Sample Registration
Samples were registered according to instructions received.
-
1-36
Plant Preparation (Dry)
Oven dried at 62°C overnight. Analytical results are reported from this
sample fraction and are not corrected for residual moisture (typically
5%), unless units denoted as %DM.
-
1-36
Nitrogen
Estimated by NIR, calibration based on N by Dumas combustion.
Result not corrected for residual moisture (typically 5%).
0.1 %
1-36
Crude Protein
Nitrogen multiplied by 6.25. Reported on DM basis.
0.5 %DM
1-36
0.5%
1-36
0.5%
1-36
0.5%
1-36
0.1 %
1-36
0.5%
1-36
0.5 MJ/kgDM
1-36
0.5%
1-36
0.5%
1-36
Acid Detergent Fibre
Neutral Detergent Fibre
Ash
Organic Matter Digestibility (in-vitro)
Digestibility of Organic Matter in Dry
Matter (DOMD)
Metabolisable Energy
Soluble Sugars
Starch
Estimated by NIR (calibration based on ADF by a modified NFTA
method). Reported on DM basis
Estimated by NIR, calibration based on NDF by NFTA method.
Reported on DM basis
Estimated by NIR, calibration based on weight loss after ashing at
600°C for two hours. Reported on DM basis.
Organic Matter Digestibility (OMD) estimated by NIR, calibration
based on AFIA (Australian Fodder Industry Association) PepsinCellulase procedure.
Calculated from Organic Matter Digestibility (OMD) using AFIA
(Australian Fodder Industry Association) Standard Equation.
Calculated from Dry Organic Matter Digestibility (DOMD) using
AFRC and Lincoln University standard formulae.
Estimated by NIR, calibration based on an 80:20 ethanol:water
extraction and colorimetric determination. Reported on DM basis.
Estimated by NIR, calibration based on Enzymic Hydrolysis of Starch.
Reported on DM basis.
Default Detection Limit
Samples
33
3. Antioxidant metabolism in White clover mosaic virusinfected Trifolium repens
3.1 Introduction
This chapter focuses on determining the extent to which the legume T. repens is able to
cope with infection induced by the Potexvirus WClMV. In particular, the effect of WClMV
was assessed in terms of plant biomass accumulation, overall fitness status, oxidative
damage and antioxidative metabolism, as well as nutritional composition of the plant.
Antioxidant markers provide a reliable indication of induced stress response in plants, and
thus a dependable measure to be utilised in the current study.
As a nitrogen-fixing plant, white clover substantially contributes to improve pastoral
quality, which is an important aspect for animal production in New Zealand (Caradus et al.,
1996). WClMV has been reported to occur at high incidence in pastures of New Zealand,
often greater than 90% (Guy and Forster, 1996). Hence, if the virus has a strong impact on
T. repens performance, the effect on yield and quality of pastures may be significant. It is
then important to understand if such detrimental effects are likely to occur in the field and
to what extent it may influence pastoral industry. Also, assessing the effect of the virus in
two widely used cultivars of white clover can bring insight on which one would suit better
for pastoral management.
3.2 Methods
The effect of WClMV infection was assessed in white clover cv. Huia and cv. Kopu II. As
mentioned in section 2.1, fifty-one seedlings of each cultivar were transplanted into
individual pots. A total of 26 replicates per cultivar were randomly selected and inoculated
with WClMV three weeks post-transplantation. Each cultivar was allocated to one bench
(as shown in Fig. 10), and the position of the pots on each bench was randomised once a
week to allow uniform growing conditions (e.g. plants placed next to the windows favoured
with more sun light). Care was taken while moving the pots to random positions to avoid
any cross-contamination.
34
Figure 10 Experimental design showing white clover Kopu II (top) and Huia (bottom) (M. Corral).
In Experiment 1, three harvests were carried out for which the whole above-ground plant
materials was cut off in a way that simulated natural grazing. After each plant had been
harvested, scissors were plunged in a bath containing soap and hot water and rinsed with
hot water only to avoid any viral contamination between uninfected and infected plants. For
each plant sample, the total above-ground material was weighed, from which about half
was frozen in liquid nitrogen and stored at -80°C prior to biochemical analysis, and the
remaining was dried for 3 days at 50°C for nutritional analysis. The initial harvest was
taken 4 weeks post-inoculation. Prior to harvests 2 and 3, plants were allowed to regrow for
a period of 3 weeks between each harvest. To assess overall plant stress, PAM
measurements were conducted fortnightly, including the days of harvesting. For each
cultivar, 5 replicates per treatment, i.e. uninfected vs infected, were randomly selected, for
which Fv/Fm values were determined from 3 leaves and subsequently averaged.
35
Below is a summary of the harvests and chlorophyll measurements undertaken over the
time of the experiment:
WClMV
inoculation
PAM 1
2 weeks
Harvest 1
PAM 2
2 weeks
PAM 3
2 weeks
Harvest 2
PAM 4
1 week
PAM 5
2 weeks
Harvest 3
PAM 6
1 week
Figure 11 Timeline showing PAM measurements and harvests of Experiment 1. Three harvests were
conducted through the study; the first one started 4 weeks post-inoculation (wpi) and the second and final
ones occurred at 7 wpi and 9 wpi, respectively (plants were allowed to regenerate for 3 weeks between each
harvest). Fv/Fm values were measured on 5 replicates per treatment (control vs WClMV-infected) every two
weeks following WClMV inoculation.
3.3 Results
3.3.1
Phenotypic variability in symptoms
T. repens exhibit high genotypic variation in fitness-related traits, such as number of
ramets/stolons, number of flowers, and total biomass. Variability in other morphological
traits, including leaf size, foliage pattern and root system, is frequently observed within
different varieties (Fig. 12). T. repens genotypes may phenotypically respond in a different
way towards WClMV infection. For instance, some were shown to be significantly
impacted in branch development, while others did not show any negative effect (Fig. 13).
Similar results from other studies have been observed for biomass production parameters
(Dzene et al., 2010; Van Molken and Stuefer, 2011). In Experiment 1, plants displayed
growth variability in response to infection, with, overall, a stunting effect induced by the
virus. In Figs. 13 & 14, the same individuals prior to Harvest 1 & 3 are compared. At 3
weeks post-inoculation (wpi), virus-free plants had grown well, while the majority of
WClMV-infected clovers had reduced size with shorter stolons. Prior to the final harvest, a
noticeable effect of previous harvesting was observed in both uninfected and infected
plants, likely due to the fact that plants were allowed to recover only for 3 weeks between
each cutting. In general, however, infected plants were dramatically affected and showed
strong stunting effect with stronger effects observed at the end of the experiment.
36
a
b
c
d
WClMV-
WClMV+
Figure 12 Phenotypic variability in uninfected and WClMV-infected leaves of white clover Huia (a and c)
and Kopu II (b and d). Leaves were collected towards the end of Experiment 1 (M. Corral).
37
Huia
Kopu II
a
d
b
e
c
WClMV-
WClMV-
WClMV+
WClMV+
Figure 13 Cultivars Huia (left) and Kopu II (right) displaying phenotypic differences (i.e. stolons
size/branching) between treatments (i.e. Uninfected vs Infected) at 3 wpi (M. Corral). Some infected plants
showed conspicuous stunted phenotypes (a,b, and d) whereas others did not appear different from control
plants (c and e).
38
Huia
Kopu II
a
d
b
e
c
WClMV-
WClMV-
WClMV+
WClMV+
Figure 14 Cultivars Huia (left) and Kopu II (right) displaying lessened phenotypic differences (i.e. stolons
size/branching) between treatments (i.e. Uninfected vs Infected) at 9 wpi (M. Corral).
39
3.3.2
Effect of WClMV infection on growth parameters
3.3.2.1 Above-ground fresh weight
16
Mean Fresh Weight
(g per plant)
Uninfected
WClMV
12
8
*
**
4
***
0
Harvest 1
Harvest 2
Harvest 3
Huia
Harvest 1
Harvest 2
Harvest 3
Kopu II
Figure 15 Mean above-ground fresh weight of uninfected (□) and WClMV-infected (■) white clover Huia
(left) and Kopu II (right) at each harvest (Huia, n = 51 / Kopu II, n = 50). Error bars indicate the standard
error of the mean for each treatment. Asterisks represent significant differences according to Student’s t-test
at P < 0.05 (*), P < 0.01 (**) and P < 0.001 (***).
The effect of WClMV infection on white clover growth was investigated in Huia and Kopu
II through the three harvests. Treatment and time (i.e. harvest) were shown to significantly
affect fresh weight (FW) in both cultivars (P < 0.01 & P < 0.001, respectively). Differences
were also found between cultivars (P < 0.001). Harvesting had a strong impact on plant
growth, yielding smaller individuals throughout the experiment. Uninfected and WClMVinfected plants were compared within each harvest so that the effect of the virus alone is
displayed. WClMV infection induced a significant decrease (30%) in plant biomass of
Huia, when compared to controls, only at the last harvest (P = 0.044). In Kopu II, virus
treatment affected the plants at an earlier stage of the experiment, with a 28% and 45%
reduction in biomass accumulation, at harvest 2 (P = 0.002) & harvest 3 (P < 0.001)
respectively (Fig. 15).
40
16
Huia
Kopu II
Mean Fresh Weight
(g per plant)
**
12
***
8
***
***
**
4
0
Harvest 1 Harvest 2 Harvest 3 Harvest 1 Harvest 2 Harvest 3
Uninfected
WClMV
Figure 16 Mean above-ground fresh weight of white clover Huia (□) and Kopu II (■) within treatments at
each harvest (Huia, n = 51 / Kopu II, n = 50). Same data as for Figure 15. Error bars indicate the standard
error of the mean for each treatment. Asterisks represent significant differences according to Student’s t-test
at P < 0.01 (**) and P < 0.001 (***).
The graph above shows the differences in biomass accumulation between the two cultivars
in response to harvesting and virus treatment. In both control and WClMV treatment, Kopu
II was significantly smaller than Huia. For uninfected plants, size differences between
cultivars were first observed at harvest 2, whereas infected Kopu II plants were already
significantly smaller than Huia at harvest 1 (Fig. 16).
41
3.3.2.2 Dry matter content
Table 2 % Dry matter in uninfected and WClMV-infected T. repens. Data is given as mean ± SD. Significant
differences between harvests were deduced from Student’s t-test and are represented by different letters.
Huia
Control
WClMV
Kopu II
Control
WClMV
Harvest 1
Harvest 2
Harvest 3
16.3 ± 0.02a
15.7 ± 0.02a
10.2 ± 0.01b
10.5 ± 0.01b
11 ± 0.01c
11.5 ± 0.01c
13.2 ± 0.02a
12.9 ± 0.01a
9.6 ± 0.01b
10.1 ± 0.01b
10.3 ± 0.01c
10.3 ± 0.01b
The percentage of dry matter (% DM) was not affected by virus treatment, although an
effect of harvest and cultivar were found (P < 0.001). % DM significantly decreased
through the time experiment and was on average lower in cultivar Kopu II (Table 2).
Mean Dry Matter Content
(g per plant)
2.5
Uninfected
WClMV
2
1.5
1
0.5
**
0
Harvest 1 Harvest 2 Harvest 3 Harvest 1 Harvest 2 Harvest 3
Huia
Kopu II
Figure 17 Dry matter content of above-ground biomass of uninfected (□) and WClMV-infected (■) white
clover Huia (left) and Kopu II (right) at each harvest. Values are given as means with error bars indicating the
standard error of the mean for each treatment. Asterisks represent significant differences according to
Student’s t-test at P < 0.01 (**).
42
According to ANOVA, a harvest and cultivar effect were found to affect dry matter content
(DMC) (P < 0.001), but no such effect were attributed to virus treatment. However, when ttests were applied, a significant difference between treatments were found in Kopu II at the
final harvest, in which infected plants displayed a 43% reduction in DMC when compared
to uninfected plants. Although no differences were highlighted in the other groups, the
trends in DMC accumulation through the harvests seem to be relatively similar to the ones
found in FW accumulation (Fig. 17).
3.3.2.1 Photosynthetic efficiency
Uninfected
Photosynthetic efficiency
(Fv/Fm)
0.80
WClMV
0.76
0.72
0.68
0.64
0.60
Huia
Kopu II
Figure 18 Effect of WClMV on photosynthetic efficiency (Fv/Fm) in Huia and Kopu II (data are given as
mean value). Error bars indicate the standard error of the mean for each treatment.
The ratio Fv/Fm estimates the maximum quantum yield of PSII, in other words, the
maximum capacity of PSII reaction centre to reduce the plastoquinone pool (QA). In
response to biotic and abiotic stresses, Fv/Fm is often seen to decrease. Accordingly, the
determination of Fv/Fm is usually used as a proxy of plant stress level (Baker, 2008). In this
experiment, no significant differences between virus treatments were found in Fv/Fm values
in each cultivar (Fig. 18).
43
3.3.3
Markers of oxidative stress
3.3.3.1 Protein carbonyls
Uninfected
WClMV
***
14
***
12
Protein carbonyls
(nmol.mg-1 protein)
***
10
8
6
4
2
0
Harvest 1 Harvest 2 Harvest 3 Harvest 1 Harvest 2 Harvest 3
Huia
Kopu II
Figure 19 Mean protein carbonyl content in uninfected (□) and WClMV-infected (■) Huia (left) and Kopu II
(right) at each harvest. Error bars indicate the standard error of the mean for each treatment. Asterisks
represent significant differences according to Student’s t-test at P < 0.001 (***).
Protein carbonylation was significantly increased in infected plants at harvest 3 only in
Huia (44% increase, P < 0.001) and at harvests 2 & 3 in Kopu II (55% increase, P < 0.001)
(Fig. 19). A time effect was found to significantly enhance protein carbonylation through
the harvests (P < 0.001), and a cultivar effect showed that Kopu II had higher protein
carbonyl contents in both uninfected and WClMV-infected plants than in Huia plants (P <
0.001).
44
3.3.3.2 Lipid peroxides
Uninfected
WClMV
30
***
***
Lipid Peroxides
(nmol.g-1 FW)
***
20
10
0
Harvest 1 Harvest 2 Harvest 3 Harvest 1 Harvest 2 Harvest 3
Huia
Kopu II
Figure 20 Levels of lipid peroxide in uninfected (□) and WClMV-infected (■) Huia (left) and Kopu II (right)
at each harvest. Error bars indicate the standard error of the mean for each treatment. Asterisks represent
significant differences according to Student’s t-test at P < 0.001 (***).
The peroxidation of lipids was significantly enhanced in virus-infected plants in the two
cultivars. In Huia plants, differences were only observed at the final harvest (~50%
increase, P < 0.001), whereas infected Kopu II plants had greater lipid peroxide contents at
harvests 2 & 3 (1.6-fold increase, P < 0.001) when compared to controls (Fig. 20). The
harvests had a significant effect on lipid peroxides, showing enhanced accumulation
through time (P < 0.001). Similar to that observed with protein carbonylation, Kopu II
showed higher lipid peroxide contents in both uninfected and infected plants than in Huia
(P < 0.001).
45
3.3.4
Non-enzymatic antioxidants
3.3.4.1 Total ascorbate levels
Uninfected
WClMV
120
***
***
***
Total Ascorbate
(µmol.g-1 FW)
100
80
60
40
20
0
Harvest 1 Harvest 2 Harvest 3 Harvest 1 Harvest 2 Harvest 3
Huia
Kopu II
Figure 21 Total ascorbate in uninfected (□) and WClMV-infected (■) Huia (left) and Kopu II (right) at each
harvest. Error bars indicate the standard error of the mean for each treatment. Asterisks represent significant
differences according to Student’s t-test at P < 0.001 (***).
Significant changes in total ascorbate levels between treatments were observed in both
cultivars. In Huia, ascorbate was 1.5-fold greater in infected plants as compared to controls
only at harvest 3 (P < 0.001). A similar increase was observed in Kopu II plants but it
occurred at an earlier stage of the experiment, i.e. harvest 2, and remained constant
thereafter (P < 0.001) (Fig. 21). A time and a cultivar effect were also correlated to the
observed differences in ascorbate content, the latter being greater in uninfected (P = 0.025)
and infected (P = 0.001) Kopu II plants when compared to Huia plants.
46
Uninfected
WClMV
% Reduced Ascorbate
100
75
50
Harvest 1
Harvest 2
Huia
Harvest 3
Harvest 1
Harvest 2
Harvest 3
Kopu II
Figure 22 % Reduced ascorbate in uninfected (□) and WClMV-infected (■) Huia (left) and Kopu II (right) at
each harvest. Error bars indicate the standard error of the mean for each treatment.
Neither time, nor virus treatment had an effect on the proportion of reduced ascorbate in the
two cultivars (Fig. 22). However, both uninfected and WClMV-infected Kopu II plants had
on average lower reduced ascorbate concentrations as compared to Huia plants (P < 0.001).
47
3.3.4.2 Total glutathione levels
Uninfected
WClMV
300
***
***
Total Glutathione
(nmol.g-1 FW)
250
***
200
150
*
100
50
0
Harvest 1 Harvest 2 Harvest 3 Harvest 1 Harvest 2 Harvest 3
Huia
Kopu II
Figure 23 Total glutathione in uninfected (□) and WClMV-infected (■) Huia (left) and Kopu II (right) at each
harvest. Error bars indicate the standard error of the mean for each treatment. Asterisks represent significant
differences according to Student’s t-test at P < 0.05 (*) and P < 0.001 (***).
Total glutathione was significantly affected by virus treatment in both cultivars. A 45%
increase in infected plants’ glutathione levels occurred in Huia at the final harvest of the
experiment (P < 0.001). In contrast, Kopu II revealed higher glutathione contents in
infected plants vs controls at harvest 1 (slight increase, P = 0.043), and harvests 2 & 3 (1.5fold increase, P < 0.001) (Fig. 23). Likewise, time (i.e. harvest) had a significant effect on
glutathione contents of the two cultivars (P < 0.001). Genotypic differences were also
observed, Kopu II plants having greater glutathione contents in both uninfected and
infected plants when compared to Huia (P < 0.001).
48
Uninfected
WClMV
% Reduced Glutathione
100
75
50
Harvest 1
Harvest 2
Huia
Harvest 3
Harvest 1
Harvest 2
Harvest 3
Kopu II
Figure 24 % Reduced glutathione in uninfected (□) and WClMV-infected (■) Huia (left) and Kopu II (right)
at each harvest. Error bars indicate the standard error of the mean for each treatment.
Similar to that of reduced ascorbate, the proportion of reduced glutathione (GSH) was not
affected by harvest or treatment (Fig. 24), and no significant differences were found
between cultivars.
49
3.3.5
Enzymatic antioxidants
3.3.5.1 Superoxide dismutase
Uninfected
WClMV
SOD Activity
(units.min-1.mg-1 protein)
250
***
200
***
***
150
100
50
0
Harvest 1 Harvest 2 Harvest 3 Harvest 1 Harvest 2 Harvest 3
Huia
Kopu II
Figure 25 Mean superoxide dismutase activity in uninfected (□) and WClMV-infected (■) Huia and Kopu II
at each harvest. Error bars indicate the standard error of the mean for each treatment. Asterisks represent
significant differences according to Student’s t-test at P < 0.001 (***).
SOD activity was affected by virus treatment in both cultivars, with increased activity in
WClMV-infected plants at the final harvest in Huia (1.4-fold greater, P < 0.001), and at
harvests 2 & 3 in Kopu II (45% and 53% increase respectively, P < 0.001) (Fig. 25). There
was also a significant effect of time (P < 0.001) on enhanced activity of the enzyme.
Differences between cultivars were also observed, with higher SOD activity being
displayed in Kopu II, when compared to Huia, in both uninfected and virus-infected plants.
50
3.3.5.2 Ascorbate peroxidase
APX Activity
(µmol. min-1.mg-1 protein)
1.6
Uninfected
WClMV
***
***
***
1.2
0.8
0.4
0
Harvest 1 Harvest 2 Harvest 3 Harvest 1 Harvest 2 Harvest 3
Huia
Kopu II
Figure 26 Mean ascorbate peroxidase activity in uninfected (□) and WClMV-infected (■) Huia and Kopu II
at each harvest. Error bars indicate the standard error of the mean for each treatment. Asterisks represent
significant differences according to Student’s t-test at P < 0.001 (***).
The activity of ascorbate peroxidase (APX) was significantly increased in WClMV-infected
Huia at harvest 3 (1.5 times higher, P < 0.001) and Kopu II at harvests 2 & 3 (1.75-fold
increase, P < 0.001) (Fig. 26). There was also a significant effect of time (P < 0.001) and
cultivar (P < 0.001), the former being positively correlated with increased enzymatic
activity and the latter showing greater APX activity in uninfected and WClMV-infected
Kopu II as compared to Huia.
51
3.3.5.3 Glutathione peroxidase
Uninfected
WClMV
***
GPX Activity
(nmol. min-1.mg-1 protein)
500
***
***
400
300
200
100
0
Harvest 1 Harvest 2 Harvest 3 Harvest 1 Harvest 2 Harvest 3
Huia
Kopu II
Figure 27 Mean glutathione peroxidase activity in uninfected (□) and WClMV-infected (■) Huia and Kopu II
at each harvest. Error bars indicate the standard error of the mean for each treatment. Asterisks represent
significant differences according to Student’s t-test at P < 0.001 (***).
The activity of GPX was significantly affected by WClMV infection. In Huia, GPX activity
was 1.5-fold greater in infected plants at harvest 3 when compared to controls. In Kopu II,
similar increases were observed at harvests 2 & 3 (Fig. 27). Significant effects of time and
cultivar (both at P < 0.001) were found. Once again, uninfected and infected Kopu II plants
had greater GPX activity than that of Huia plants.
52
3.3.5.4 Catalase
CAT Activity
(µmol. min-1.mg-1 protein)
350
Uninfected
WClMV
***
300
***
***
250
200
150
100
50
0
Harvest 1 Harvest 2 Harvest 3 Harvest 1 Harvest 2 Harvest 3
Huia
Kopu II
Figure 28 Mean catalase activity in uninfected (□) and WClMV-infected (■) Huia and Kopu II at each
harvest. Error bars indicate the standard error of the mean for each treatment. Asterisks represent significant
differences according to Student’s t-test at P < 0.001 (***).
Catalase activity was markedly increased in WClMV-infected plants in both Huia and
Kopu II (of about 1.5-fold times), at harvest 3 and harvests 2 & 3 respectively (P < 0.001)
(Fig. 28). A time effect was greatly correlated to increased CAT activity in each cultivar
(P < 0.001). There were also significant differences between cultivars, with higher CAT
activity observed in control and virus-treated Kopu II plants when compared to Huia.
53
3.3.5.5 Glutathione S-transferase
GST Activity
(nmol DNPG.min-1.mg-1 protein)
Uninfected
WClMV
50
40
30
20
10
0
Harvest 1 Harvest 2 Harvest 3 Harvest 1 Harvest 2 Harvest 3
Huia
Kopu II
Figure 29 Mean glutathione S-transferase activity in uninfected (□) and WClMV-infected (■) Huia and Kopu
II at each harvest. Error bars indicate the standard error of the mean for each treatment.
Glutathione S-transferase activity did not differ between treatments or between harvests
(Fig. 29). Significant differences were only displayed between cultivars. Indeed, levels of
GST were higher in uninfected and virus-infected Kopu II plants in comparison with Huia
plants (P < 0.001).
54
3.3.5.6 Glutathione reductase
Uninfected
WClMV
***
GR Activity
(nmol.min-1.mg-1 protein)
250
***
***
200
150
100
50
0
Harvest 1 Harvest 2 Harvest 3 Harvest 1 Harvest 2 Harvest 3
Huia
Kopu II
Figure 30 Mean glutathione reductase activity in uninfected (□) and WClMV-infected (■) Huia and Kopu II
at each harvest. Error bars indicate the standard error of the mean for each treatment. Asterisks represent
significant differences according to Student’s t-test at P < 0.001 (***).
Glutathione reductase activity was significantly affected by virus treatment. WClMVinfected Huia plants showed a 40% increase in GR activity at harvest 3 (P < 0.001), while
virus-treated Kopu II plants displayed enhanced activity at harvests 2 & 3 (~1.6 times
increase, P < 0.001) (Fig. 30). A harvest effect was significantly correlated to induced GR
activity in the two cultivars (P < 0.001). Differences in the levels of GR were also
displayed between cultivars, with greater enzymatic activity in uninfected and virus-treated
Kopu II plants when compared to Huia (P < 0.001).
55
Table 3 Summary of ANOVA results for growth parameters, oxidative damage and antioxidant activities measured in Experiment 1.
Treatmenta
Growth parameters
Above-ground fresh weight
% Dry matter
Dry matter content
Photosynthetic efficiency (Fv/Fm)
Markers of oxidative stress
Protein carbonyl content
Lipid peroxide content
Non-enzymatic antioxidants
Total ascorbate activity
Reduced ascorbate activity
Total glutathione activity
Reduced glutathione activity
Enzymatic antioxidants
Superoxide dismutase activity
Ascorbate peroxidase activity
Glutathione peroxidase activity
Catalase activity
Glutathione S-transferase
Glutathione reductase
a
Treatment: Uninfected / WClMV
b
Time: Harvest 1 - 2 - 3
c
Genotype: Huia / Kopu II
*
Fv/Fm values measured throughout the experiment
Timeb
Genotypec
F1, 291 = 9.20, P < 0.01
F2, 291 = 177.45, P < 0.001
F1, 291 = 54.42, P < 0.001
F1, 263 = 0.22, P > 0.05
F2, 263 = 337.19, P < 0.001
F1, 263 = 85.45, P < 0.001
F1, 263 = 2.31, P > 0.05
F2, 263 = 188.72, P < 0.001
F1, 263 = 53.65, P < 0.001
*
F1, 96 = 0.62, P > 0.05
F5, 96 = 2.12, P > 0.05
F1, 96 = 0.01, P > 0.05
F1, 192 = 171.01, P < 0.001
F2, 192 = 310.83, P < 0.001
F1, 192 = 200.49, P < 0.001
F1, 192 = 207.63, P < 0.001
F2, 192 = 330.97, P < 0.001
F1, 192 = 264.23, P < 0.001
F1, 192 = 175.18, P < 0.001
F2, 192 = 328.02, P < 0.001
F1, 192 = 90.60, P < 0.001
F1, 192 = 2.05, P > 0.05
F2, 192 = 0.86, P > 0.05
F1, 192 = 35.77, P < 0.001
F1, 192 = 128.40, P < 0.001
F2, 192 = 250.80, P < 0.001
F1, 192 = 206.01, P < 0.001
F1, 192 = 0.25, P > 0.05
F2, 192 = 0.21, P > 0.05
F1, 192 = 0.15, P > 0.05
F1, 192 = 155.83, P < 0.001
F2, 192 = 331.59, P < 0.001
F1, 192 = 248.60, P < 0.001
F1, 192 = 186.14, P < 0.001
F2, 192 = 306.01, P < 0.001
F1, 192 = 206.01, P < 0.001
F1, 192 = 148.68, P < 0.001
F2, 192 = 239.37, P < 0.001
F1, 192 = 162.31, P < 0.001
F1, 192 = 191.50, P < 0.001
F2, 192 = 288.26, P < 0.001
F1, 192 = 248.75, P < 0.001
F1, 192 = 0.57, P > 0.05
F2, 192 = 0.50, P > 0.05
F1, 192 = 71.60, P < 0.001
F1, 192 = 142.36, P < 0.001
F2, 192 = 297.93, P < 0.001
F1, 192 = 135.85, P < 0.001
56
3.3.6
Nutrient analyses
3.3.6.1 Nitrogen and ash content
Table 4 Content of nitrogen and ash in T. repens Huia & Kopu II at each harvest. Data represent the mean ±
SD from three replicates. Groups were compared using generalized linear models (GLM) with Bonferroni
post-hoc analysis. Values with different letters are significantly different. DM = dry matter.
Huia
Kopu II
Harvest 1
Harvest 2
Harvest 3
Harvest 1
Harvest 2
Harvest 3
2.70 ± 0.30a
4.77 ± 0.32b
4.73 ± 0.35b
3.50 ± 0.50a
5.30 ± 0.56b
5.10 ± 0.20b
2.47 ± 0.25a
5.10 ± 0.40b
4.63 ± 0.15b
3.63 ± 0.15a
5.20 ± 0.26b
5.17 ± 0.12b
8.20 ± 0.79a
12.83 ± 1.29b
10.67 ± 0.81c
8.97 ± 0.67a
12.13 ± 0.35b
11.37 ± 0.38b
8.50 ± 0.40a
12.40 ± 0.10b
10.57 ± 0.12c
9.23 ± 0.23a
11.90 ± 0.78b
11.17 ± 0.12b
Nitrogen (% DM)
Control
X±S.D.
WClMV
X±S.D.
Ash (% DM)
Control
X±S.D.
WClMV
X±S.D.
Nitrogen and ash contents were significantly affected by time (P < 0.001), but not by
treatment, in both cultivars. Differences between cultivars were only found in nitrogen
concentration (P < 0.001), being slightly higher in Kopu II. Over the time of the
experiment, nitrogen contents increased by about 80% and 40%, and ash contents increased
by about 40% and 25% in Huia and Kopu II, respectively (Table 4).
57
3.3.6.2 Crude protein and fibre content
Table 5 Content of crude protein (CP), acid detergent fibre (ADF) and neutral detergent fibre (NDF) in T.
repens Huia & Kopu II at each harvest. Data represent the mean ± SD from three replicates. Groups were
compared using generalized linear models (GLM) with Bonferroni post-hoc analysis. Values with different
letters are significantly different. DM = dry matter.
Huia
Kopu II
Harvest 1
Harvest 2
Harvest 3
Harvest 1
Harvest 2
Harvest 3
18.87 ± 2.21a
32.30 ± 2.12b
32.60 ± 2.31b
24.50 ± 3.55a
36.30 ± 3.77b
35.10 ± 1.57b
17.27 ± 1.93a
35.00 ± 2.75b
31.67 ± 0.76b
25.03 ± 1.31a
35.40 ± 1.95b
35.47 ± 0.81b
11.33 ± 1.85a
20.87 ± 0.55b
17.10 ± 0.44c
15.07 ± 0.23a
18.40 ± 0.36b
17.07 ± 0.75ab
13.53 ± 1.65a
18.07 ± 0.87b
16.53 ± 0.51b
16.97 ± 0.68
18.83 ± 0.42
17.90 ± 1.31
13.63 ± 2.40a
21.07 ± 0.23b
17.50 ± 1.42c
17.27 ± 1.00
18.57 ± 1.10
16.80 ± 0.52
15.83 ± 1.50
18.13 ± 1.29
16.73 ± 0.75
17.13 ± 1.60
18.67 ± 1.00
17.47 ± 1.46
CP (% DM)
Control
X±S.D.
WClMV
X±S.D.
ADF (% DM)
Control
X±S.D.
WClMV
X±S.D.
NDF (% DM)
Control
X±S.D.
WClMV
X±S.D.
In both cultivars, contents of crude protein (CP), acid detergent fibre (ADF) and neutral
detergent fibre (NDF) were affected significantly by time (P < 0.001), but not by virus
treatment. Cultivar effects were only found in CP and ADF contents (P < 0.001 & P <
0.01, respectively), the latter being greater in Kopu II as compared to Huia. On average, CP
content increased by 1.8-fold in Huia and 1.4-fold in Kopu II. ADF content in Huia was
about 45% greater at the end of the experiment, whereas Kopu II displayed a small increase
(13%) only in controls as WClMV-infected plants did not significantly differ. Changes in
NDF content were only found to be significant in virus-free Huia plants, displaying a 40%
increase (Table 5).
58
3.3.6.3 Soluble sugars and starch content
Table 6 Content of soluble sugars and starch in T. repens Huia & Kopu II at each harvest. Data represent the
mean ± SD from three replicates. Groups were compared using generalized linear models (GLM) with
Bonferroni post-hoc analysis. Values with different letters are significantly different. DM = dry matter.
Huia
Kopu II
Harvest 1
Harvest 2
Harvest 3
Harvest 1
Harvest 2
Harvest 3
15.90 ± 0.36a
7.17 ± 1.65b
11.30 ± 6.53c
13.53 ± 1.68a
6.20 ± 0.87b
9.10 ± 0.87c
17.10 ± 1.57a
7.07 ± 0.21b
11.67 ± 0.71c
12.93 ± 1.59a
5.63 ± 0.81b
8.93 ± 0.60c
2.63 ± 2.75
< 0.5
< 0.5
< 0.5
< 0.5
< 0.5
1.33 ± 0.50
< 0.5
< 0.5
< 0.5
< 0.5
< 0.5
Soluble Sugars
(% DM)
Control
X±S.D.
WClMV
X±S.D.
Starch (% DM)
Control
X±S.D.
WClMV
X±S.D.
In both cultivars, soluble sugar content was significantly affected by time and cultivar
(both, P < 0.001), but not by virus treatment. Statistical analyses could not be performed for
starch content as reported values were too small, hence considered not reliable for
interpretation. Over the time of the experiment, the contents of soluble sugars in both
cultivars dropped by about 45%. Our results also showed that Huia had significantly higher
soluble sugar content than Kopu II (Table 6).
59
3.3.6.4 Digestibility of organic matter and metabolisable energy
Table 7 Digestible organic matter in the dry matter (DOMD) and metabolisable energy (ME) in T. repens
Huia & Kopu II at each harvest. Data represent the mean ± SD from three replicates. Groups were compared
using generalized linear models (GLM) with Bonferroni post-hoc analysis. Values with different letters are
significantly different. DM = dry matter.
Huia
Kopu II
Harvest 1
Harvest 2
Harvest 3
Harvest 1
Harvest 2
Harvest 3
83.53 ± 1.26a
78.97 ± 1.59b
84.37 ± 0.21ac
82.33 ± 0.15
80.60 ± 1.48
83.73 ± 0.61
81.80 ± 0.56ab
80.50 ± 1.15a
84.23 ± 1.14b
80.67 ± 0.38
81.13 ± 0.85
82.63 ± 1.53
13.37 ± 0.15a
12.63 ± 0.23b
13.50 ± 0.00ac
13.20 ± 0.00
12.90 ± 0.26
13.40 ± 0.10
13.10 ± 0.10ab
12.87 ± 0.21a
13.47 ± 0.21b
12.93 ± 0.06
12.97 ± 0.15
13.23 ± 0.25
DOMD (%)
Control
X±S.D.
WClMV
X±S.D.
ME
(MJ.kg-1 DM)
Control
X±S.D.
WClMV
X±S.D.
The contents of digestible organic matter in the dry matter (DOMD) and metabolisable
energy (ME) were affected significantly by time only (P < 0.001). No statistical differences
were found in both parameters in Kopu II. In Huia plants, significant differences in DOMD
and ME values were found between harvests, however, results from the final harvesting did
not differ from the ones obtained at the start of the experiment (Table 7).
60
Table 8 Summary of ANOVA results for nutritional parameters measured in Experiment 2.
Nutritional parameters
Nitrogen
Ash
Crude protein
Acid detergent fibre
Neutral detergent fibre
Soluble sugars
Starch
Digestible organic matter in the dry matter
Metabolisable energy
a
Treatment: Uninfected / WClMV
b
c
Time: Harvest 1 - 2 - 3
Genotype: Huia / Kopu II
n.a. = not applicable
Treatmenta
Timeb
Genotypec
F1, 24 = 0.02, P > 0.05
F2, 24 = 141.01, P < 0.001
F1, 24 = 28.94, P < 0.001
F1, 24 = 0.11, P > 0.05
F2, 24 = 104.47, P < 0.001
F1, 24 = 1.70, P > 0.05
F1, 24 = 0.001, P > 0.05
F2, 24 = 127.63, P < 0.001
F1, 24 = 28.08, P < 0.001
F1, 24 = 1.11, P > 0.05
F2, 24 = 78.74, P < 0.001
F1, 24 = 12.88, P < 0.01
F1, 24 = 0.11, P > 0.05
F2, 24 = 17.75, P < 0.001
F1, 24 = 1.32, P > 0.05
F1, 24 = 0.01, P > 0.05
F2, 24 = 156.32, P < 0.001
F1, 24 = 37.58, P < 0.001
n.a.
n.a.
n.a.
F1, 24 = 1.54, P > 0.05
F2, 24 = 33.15, P < 0.001
F1, 24 = 1.23, P > 0.05
F1, 24 = 1.64, P > 0.05
F2, 24 = 32.81, P < 0.001
F1, 24 = 0.79, P > 0.05
61
3.4 Discussion
Throughout their life cycle, plants must continuously cope with various stress conditions,
including pathogen invasion that may lead to disease. To overcome such constraints, plants
have evolved mechanisms to sense and respond to such perturbations, often involving the
role of phytohormones and defence genes, the activation of specific signalling pathways,
and the interplay between ROS and antioxidative metabolism (Fujita et al., 2006).
In plant-virus interactions, the ability of the host to specifically recognize the intruder is a
crucial step that may strongly influence host resistance and virus propagation. Upon
successful recognition, in which a specific interaction between plant R and viral avr
proteins occurs, cascades of transduction signals are activated and lead to the initiation of
the hypersensitive response (HR), an interaction termed incompatible (Stange, 2006). The
HR takes place at the infection site where it triggers programmed cell death events (Dardick
and Culver, 1999; Pallas and García, 2011) and it is often closely related to the
development of SAR (Kombrink and Schmelzer, 2001). Plant resistance against viruses
may also involve a large set of proteins called pathogenesis-related proteins, defined as any
host proteins induced in response to pathogen infection and often associated with the
induction of SAR (Palukaitis et al., 2008). Another molecular response often induced by
viral RNA is RNA silencing, which results in the targeting of dsRNA structures, its
cleavage yielding small interfering RNAs (siRNAs) and enhancing viral ssRNA
degradation or inhibition of viral translation (Pallas and García, 2011). The Potexvirus
WClMV does not induce any HR response in white clover. Instead, symptoms appear on
the leaves and are characterized by light/dark green mosaic patterns (Fig. 9). At first glance,
this indicates that T. repens resistance mechanisms against WClMV, if any occur, are likely
to be independent of SAR induction. Broderick et al. (1997) identified a PR gene in
subterranean clover (Trifolium subterraneum) that encodes a PR protein under attack by
Halotydeus destructor (a species of mite), for which a chitinase activity was suggested. To
our knowledge, no PR genes that may be involved in WClMV resistance have yet been
identified in white clover.
62
The effect of WClMV on growth parameters and biochemistry of T. repens was assessed in
two cultivars. WClMV infection had an adverse impact on biomass accumulation in both
cultivars, inducing stunted phenotypes for the majority of infected plants. Stunting is a
common symptom induced by plant viruses, and is mainly a consequence of decreased leaf
size and reduced internode length (Hull, 2009). Our results are in accordance with previous
studies describing similar effects of WClMV on T. repens growth rate (Van Molken and
Stuefer, 2011; Woodfield and Caradus, 1996). WClMV infection reduced the total fresh
weight (FW) of white clover (Fig. 15), with Kopu II having suffered more from the virus
than Huia (Fig. 16). Differences between cultivars are not unusual, van Molken et al.
(2011) having also reported such variations in growth-related traits in response to WClMV
infection in different genotypes of white clover (Van Molken and Stuefer, 2011). Although
trends in dry matter content (DMC) accumulation between controls and infected plants
were similar to total FW, statistical analyses did not indicate significant differences, except
in Kopu II for which DMC had decreased by about 40% by the final harvest (refer to Fig.
16). However, it seems the more the plants are subjected to harvesting, the less DMC is
produced when compared to controls. Hence, it is most likely that further harvests would
have yielded infected plants with drastic reduction in DMC. Considering the high incidence
of WClMV in New Zealand’s grasslands (Guy and Forster, 1996), its impact on T. repens
yield is an important factor to assess as it may strongly affect the quality and productivity
of pastures. Fry (1959) investigated the effect of WClMV on T. repens in glasshouse trials
and reported reduction of 24% in leaf yield. Likewise, Van Molken et al. (2011) reported a
decline in total plant biomass of white clover by up to 30% under WClMV infection. In the
current study, reduction in dry matter yield caused by the virus was only observed in Kopu
II, suggesting that Huia would be more suitable for pastoral use. In addition, the percentage
of dry matter in T. repens was not affected by the virus, suggesting that WClMV infection
may have no effect of plant tissue composition.
Additionally, the ratio Fv/Fm was estimated to assess overall plant health. No significant
variation in maximum photochemical efficiency was found between controls and infected
plants, indicating that WClMV infection per se was not sufficient to induce photo-damage
in PSII reaction centres. Other studies have reported similar observations. As exemplified
by Guo et al. (2005), Fv/Fm ratio remained the same in stem mustard infected with Turnip
63
mosaic virus (TuMV) compared to healthy plants. Similar results were also observed in
PVYNTN-infected potato plants which, despite decreased net photosynthetic rate, did not
show any noticeable changes in photochemical efficiency (Zhou et al., 2004). Although
T. repens biomass accumulation has been shown to be negatively affected by WClMV
infection, its photosynthetic performance does not seem to be impacted. This indicates that,
even under infection, the plant is still able to photosynthesize.
In many plant-pathogen interactions, one of the earliest defence responses upon pathogen
recognition is the rapid and transient production of ROS (mostly O2•-, H2O2 and HO•), a
process called oxidative burst (Mendoza, 2011; Wojtaszek, 1997; Yoshioka et al., 2008).
ROS accumulation is intense during incompatible interactions, it often occurs in a biphasic
fashion and is usually correlated to resistance. Under infection with virulent pathogens, i.e.
during compatible interactions, only one phase of ROS generation is often observed (Shetty
et al., 2008). Under biotic stress, ROS formation usually involves the activity of the plasma
membrane-bound NADPH oxidase, although apoplastic production by cell-wall
peroxidases, amine oxidases and oxalate oxidases may also occur (Bolwell et al., 2002;
Bolwell and Wojtaszek, 1997). Changes in membrane permeability followed by Ca2+ and
H+ influx, and K+ and Cl- efflux have been observed upon pathogen recognition.
Intracellular Ca2+ accumulation may have a direct effect on NADPH oxidase, the latter
enhancing O2•- production. Superoxide anions are subsequently dismuted by SOD to H2O2,
of which the signalling function may be importantly involved in the induction of defence
mechanisms, including the HR. Furthermore, extracellular alkalinisation following K+ and
Cl- efflux may initiate apoplastic oxidative burst by activating cell-wall peroxidases also
involved in H2O2 production (Wojtaszek, 1997; Yoshioka et al., 2008). Although ROS may
have important roles as signalling molecules for the induction of defence mechanisms
under pathogen attack, the plant cell still must cope with their associated toxicity through
the participation of ROS-scavenging antioxidants. Therefore, it is crucial that the overall
balance between the production and the removal of ROS occurring during plant-pathogen
interaction is tightly controlled (Alscher et al., 1997; Mittler, 2002).
Biochemical analyses were performed to measure the level of oxidative damage and
activity of antioxidants triggered by virus infection. Protein carbonylation and lipid
64
peroxidation are two reliable markers of ROS-induced oxidative stress involved in
oxidation of proteins and oxidative degradation of membrane lipids, respectively (Marrs,
1996; Moller et al., 2007). WClMV infection in both cultivars induced protein oxidation
and lipid peroxidation as evidenced by significant increases in protein carbonyl and lipid
peroxide contents, indicating that oxidative damage occurred. Previous studies have also
reported similar outcomes in response to plant virus infection. In susceptible Dactylis
glomerata plants infected with Cocksfoot mottle virus (CfMV), levels of lipid peroxide
significantly rose as symptoms developed on the leaves of tillers (Li and Burritt, 2003).
Similarly, accumulation of carbonyl-proteins was observed in pea plants and peach leaves
infected with Plum pox virus (Díaz-Vivancos et al., 2008; Hernández et al., 2006). This
study indicated Kopu II was oxidatively more damaged as protein oxidation and lipid
peroxidation levels were higher and initiated earlier than in Huia. Likewise, the profiles of
oxidative stress observed in this experiment were consistent with the decreased growth
patterns of both cultivars (as shown in Figs. 15, 19 and 20), thus emphasizing the adverse
effect of virus-induced oxidative damage on white clover performance.
The non-enzymatic antioxidants ascorbate and glutathione are major redox buffers in plant
cells. They are both readily oxidized by stress-generated ROS in order to alleviate the effect
of oxidative damage (Conklin, 2001; Noctor et al., 2012; Smirnoff, 1996). Accordingly, the
ascorbate-glutathione cycle plays a central role in oxidative responses, and the enzymes
involved are likely to be stimulated under stress conditions (Zhang, 2013). In this study,
ascorbate and GSH levels were significantly increased in virus-treated plants of both
cultivars. However, no changes in the proportions of reduced ascorbate and reduced
glutathione were found between treatments. To perform greater functions as ROS
scavengers, the cell must maintain suitable levels of reduced forms of antioxidants. One
may conclude that elevations in ascorbate and GSH pools with no noticeable changes in
redox status would indicate that a rapid regeneration of reduced forms following oxidation
by ROS must have occurred in such a way that the reduced/oxidized ratios remained
constant. In both cultivars, infected plants were able to maintain high ratios of
reduced/oxidized ascorbate & GSH throughout, representing at least 75% of reduced
molecules.
65
To counteract the adverse effects of ROS produced under environmental stresses, plants
have evolved a large array of enzymatic antioxidants, including SOD, APX, GPX, CAT,
GST, and GR (Ahmad et al., 2008). Superoxide dismutases are found in all cellular
compartments and represent the first defensive barrier against ROS, whose function is to
dismute O2•- to H2O2 (Alscher et al., 2002; Apel and Hirt, 2004). SOD activity was
significantly affected by WClMV, showing increased levels in the two cultivars. Other
authors have reported comparable results. In response to CMV infection, Song et al. (2009)
found cucumber and tomato plants with higher levels of SOD in chloroplasts and
mitochondria when compared to uninfected controls. SOD activity was also induced in faba
bean leaves infected with BYMV (Radwan et al., 2010), as well as in N. benthamiana
plants infected with a virulent strain of PMMoV (Hakmaoui et al., 2012). As the activity of
SOD increased in WClMV-infected T. repens, H2O2 contents must have risen
consequently. H2O2 plays important roles in signalling cascades in response to various
abiotic and biotic stresses (Cheeseman, 2007). In particular, the involvement of H2O2 in
plant defence responses against pathogens has been extensively studied (Bestwick et al.,
1997; Chamnongpol et al., 1998; Kuzniak and Urbanek, 2000; Quan et al., 2008; Wu et al.,
1997). H2O2 has been shown to directly affect survival and viability of invaders, to enhance
cell wall reinforcement, to induce the hypersensitive response and activity of antimicrobial
compounds, and to interact with resistance-involved phytohormones (Bestwick et al., 1997;
Chamnongpol et al., 1998; Kuzniak and Urbanek, 2000; Quan et al., 2008; Wu et al., 1997).
Although H2O2 content was not assessed in this study, it is likely that levels of H2O2 were
increased in T. repens upon infection as ROS-induced oxidative damage occurred.
In Huia and Kopu II, ascorbate peroxidase and glutathione peroxidase activities were
significantly induced by infection. This is in accordance with a number of studies having
reported increased peroxidase activities in virus-infected plants (Clarke et al., 2002; DíazVivancos et al., 2006; Hakmaoui et al., 2012; Radwan et al., 2010; Riedle-Bauer, 2000;
Song et al., 2009). Both APX and GPX are important enzymatic antioxidants involved in
the reduction of H2O2 formed under oxidative damage, respectively using ascorbate and
GSH as a source of reductants (Gill and Tuteja, 2010). The results indicate that WClMVinfected white clover can effectively trigger the activity of ROS-scavenging peroxidases to
cope with virus-induced oxidative stress. This would imply sufficient availability in
66
reduced forms of ascorbate and glutathione for efficient peroxidase activity. As previously
mentioned, ascorbate and glutathione pools were drastically enhanced in infected plants and
were maintained at high redox status. This indicates that APX and GPX would have been
supplied with sufficient amount of reduced substrates to detoxify H2O2.
Catalase, another enzyme involved in ROS detoxification, is responsible for the reduction
of H2O2 to water (Scandalios et al., 1997). Its activity significantly increased by at least
50% in infected plants of both cultivars. Similar observations have been reported in faba
bean infected with BYMV (Radwan et al., 2010), as well as in CMV- & ZYMV-infected
Cucumis sativus and Cucurbita pepo plants (Riedle-Bauer, 2000). In this study, the results
indicate that, in response to WClMV infection, T. repens is able to enhance CAT activity to
counteract stress-induced oxidative damage.
Importantly involved in the ascorbate-glutathione cycle, glutathione reductase also acts in
preventing oxidative damage, although not directly, by regenerating GSH (Ding et al.,
2009). Enhancement in GR activity was observed in infected plants of both cultivars.
Hence, GSSG must have been effectively reduced to GSH, thus maintaining the ratio
reduced/oxidized glutathione at a high level (which is consistent with our results mentioned
previously).
Interestingly, WClMV infection did not have any effect on glutathione S-transferase
activity in both cultivars when compared to controls. The main role of GSTs is to facilitate
the conjugation of GSH to electrophilic xenobiotic substrates often toxic, hence preventing
cellular damage (Dixon et al., 2010; Marrs, 1996). Our results indicate that, in WClMVtreated T. repens, GST do not participate in ROS-scavenging mechanisms or, if so, its
participation may not be predominant.
Collectively, the biochemical results indicate that oxidative damage with generation of
ROS was induced by WClMV infection in T. repens, as highlighted by increased lipid
peroxidation and protein carbonylation. To alleviate the effect of activated oxygen, white
clover effectively triggered ROS-scavenging mechanisms by enhancing the availability of
ascorbate and glutathione, increasing the activities of enzymatic antioxidants, as well as
67
sustaining high cellular redox potential. Enhanced SOD activity must have strongly
participated in the detoxification of highly reactive superoxide anions to less toxic H2O2,
the latter being subsequently reduced by CAT, APX and GPX, whose activities were also
increased. Sufficient levels and higher availability of reduced forms of ascorbate and
glutathione were closely correlated with enhanced activity of APX, GPX and GR. The
changes in antioxidant metabolism observed here are likely to have conferred to the plant
greater chance to alleviate virus-induced oxidative damage. Hence, from a biochemical
aspect, it appears that T. repens is capable of coping with the Potexvirus WClMV.
Feed quality analyses provided information on the nutritive value of T. repens grown for
about 6 months, infected with WClMV and prone to three harvests. The results showed that
WClMV had no effect on all measured nutritional parameters, but differences between the
two genotypes were observed. Cultivar Kopu II had higher contents in nitrogen, crude
protein and acid detergent fibre than Huia. However, the latter had a higher soluble sugar
content. Overall, in both cultivars, nitrogen, ash, CP and ADF contents significantly
increased over the time experiment, a slight increase in neutral detergent fibre were only
seen in Huia plants. In contrast, although virus infection was often reported to increase the
levels of sugars in plants (Moghaddam and Van den Ende, 2012), content in soluble sugars
decreased at the same rate in both cultivars. It is likely that these changes are a consequence
of plant maturation and/or harvesting-induced stress. In addition, the values of digestible
organic matter and metabolisable energy did not differ throughout the harvests. The results
of increased nitrogen were surprising, instead N content would have been expected to
decline in virus-treated plants, as it has been reported that N2-fixation ability was impaired
in WClMV-infected white clover (Dudas et al., 1998; Guy et al., 1980) as well as in other
plant-virus systems (Khadhair and Sinha, 1984; Wroth et al., 1993). In this study, clover
plants were still able to assimilate nitrogen even under infection or harvesting-induced
stress. Likewise, ash content, or total mineral content, in both cultivars remained unchanged
between virus treatment but increased throughout time, and was closely similar to some
reported in other studies (Berardo, 1997; Harrington et al., 2006). The following
interpretations are based on online reports provided by R J Hill Laboratories Limited
(Hamilton, New Zealand). ADF and NDF, mainly consisting of cellulose and lignin (plus
hemicelluloses for NDF) measure the fibre content in forages. High levels of fibre should
68
be avoided for livestock feeding as it slows the rate of digestion, although a certain amount
must be available for optimal rumen activity. However, very low levels (as in the present
study) may be related to metabolic dysfunctions. It is important to keep in mind that in the
present case, white clover plants were only grown for a limited period of time and may
have not properly matured, which would explain the lack of fibre. Crude protein contents
were high in both Huia and Kopu II, even when infected by the virus, and satisfactory for
silage productivity. Digestible organic matter and metabolisable energy did not show any
major variations over the time experiment. However, both were present in T. repens at high
levels, indicating excellent status for nutrient intake and high amount of energy available
for livestock. Soluble sugars, also called water-soluble carbohydrates, are important for the
diet of ruminants as they stimulate/maintain the microflora activity for efficient digestion
process. The levels of soluble sugars measured in this study were moderate in both
cultivars, ranging from 7.07 to 17.1 % DM in Huia and from 5.63 to 13.53 % DM in Kopu
II, significant decreases having been observed by the end of the experiment.
Collectively, these results indicate that WClMV infection induces oxidative damage in T.
repens, as evidenced by enhanced protein carbonylation and lipid peroxidation (markers of
oxidative stress). Total ascorbate and total glutathione pools were higher in infected
clovers, although no changes in reduced/oxidized ratio occurred, most likely as a mean of
maintaining a reduced state to prevent harmful effects of ROS. Except for GST, the
enzymatic activities of SOD, APX, GPX, CAT and GR were increased in WClMV-infected
plants. This suggests that under infection with WClMV, T. repens can trigger defence
mechanisms to alleviate intracellular oxidative damage and cope with the virus.
Interestingly, the kinetics of all non-enzymatic and enzymatic antioxidants (with the
exception of GST) were consistent with the occurrence of oxidative stress and the trends in
decreased plant biomass, in both cultivars. In addition, the harvesting per se had a
significant effect on biomass accumulation, markers of oxidative stress as well as on most
antioxidants. After defoliation, regrowth and maintenance of plant biomass depends mainly
on stored carbon (Caldwell et al., 1981), which, in T. repens is expected to be found as
carbohydrates in the stolons. It may be costly for the plant to regrow under virus infection
condition as resources are likely to be allocated towards the defence mechanisms triggered
against the virus, therefore impacting its growth performance. This is in accordance with
69
the results of the nutritional analyses indicating that soluble sugar contents were decreased
throughout the time experiment in both virus-free and infected plants (refer to Table 6).
Furthermore, it is conceivable that harvesting, which occurs extensively in natural
agrosystems via grazing animals, may itself act as an abiotic stress, and when coupled to
WClMV infection, the interactive effect of both stresses may be even more detrimental for
T. repens. However, although white clover was shown to be significantly impacted by virus
and harvest, infected plants were nutritionally as valuable as uninfected controls.
This study also demonstrated that Kopu II was more sensitive to WClMV infection as
reduction in biomass and increase in oxidative damage were greater, and also occurred
much earlier in the experiment (harvest 2), than in Huia. For the reasons mentioned above,
the current study indicates that Huia is able to cope more effectively with the virus and
would be a preferable candidate for pastoral systems over Kopu II. However, looking at the
nutritional status, Kopu II would bring benefits as it displayed higher nitrogen, mineral and
protein contents.
It is most likely that in the field, infection with viruses is not the only negative pressure that
white clover would have to cope with, many other abiotic and biotic stresses being also
involved. Excess water in soil is one of them, and may importantly affect physiology and
biochemistry of the legume. One may postulate that plants would respond differently to that
environmental perturbation when infected with WClMV. To assess this hypothesis, a
second experiment was conducted wherein a root-flooding treatment was applied to
uninfected and virus-treated white clover (refer to the next Chapter).
70
4. The effect of root-flooding on oxidative damage and
antioxidant metabolism in Trifolium repens infected or
not with WClMV
4.1 Soil flooding: an important abiotic stress
Known to significantly impact distribution and productivity of plant species worldwide
(Jackson and Colmer, 2005; Jackson et al., 2009), soil flooding often occurs as a result of
over-irrigation or heavy precipitation, being particularly intense in poorly drained soils
(Huang, 2000). One of the predictions of climate change is increased precipitation and
flooding in some regions. Studies predict heavier and/or more frequent rainfall in western
areas of New Zealand accompanied with expected increase of floods due to intense
precipitation in winter (Hollis and Mullan, 2008; New Zealand Ministry of the
Environment, 2009). Investigating how water excess may impair plant performance and
affect biotic interactions is a novel area of research that will bring interesting insights and is
of prime importance considering the likelihood of increased rainfall in the coming century.
In water, oxygen diffuses at a slower rate (about 10,000-fold) than in the air (Bailey-Serres
and Voesenek, 2008; Sairam et al., 2008). In cells, oxygen is a key element in the
mitochondrial electron transport chain where its function is crucial as the terminal electron
acceptor. Therefore, as oxygen dramatically decreases in flooded roots, mitochondrial
functions along with ATP production are disturbed. This may lead to growth inhibition,
disruption in membrane transport and cause senescence of plant tissues. However,
glycolysis and fermentative pathways can provide ATP molecules under low oxygen
availability, although the yield of ATP production is limited (Sairam et al., 2008; Teakle et
al., 2006). Oxygen deficiency may also disrupt nutrient and water uptakes and results in a
change of metabolic functions, shifting from aerobic to anaerobic systems (Sairam et al.,
2008). Shoot and root growth can be negatively affected by waterlogging, root formation
being particularly sensitive to lack of oxygen. In many cases, adventitious roots are often
formed near the surface to allow oxygenation of anoxic root parts. Waterlogging may also
impede development and growth of leaves, and induce leaf chlorosis and senescence
71
(Huang, 2000). In legume plants, flooding-induced hypoxia may adversely impact
nodulation process and nitrogen fixation due to oxygen limitation (Roberts et al., 2010).
Soil flooding exerts great selection pressure on plants that may result in different
adaptations allowing plants to tolerate that stress to some extent, depending on the degree
of occurrence of flooding. Increased root porosity, adventitious root formation, enhanced
development of petiole and shoot are traits selected in response to frequent/predictable
waterlogging conditions (Huber et al., 2009).
Under O2 depletion, as cellular energy (ATP) dramatically decreases (falling from 36 to 2
moles per mole of glucose metabolised), the cell must depend on glycolytic and
fermentative processes to generate ATP and regenerate NAD+ to further sustain anaerobic
metabolism (Bailey-Serres and Voesenek, 2008; Ferreira de Sousa and Sodek, 2002).
Ethanol and lactic acid are among the major products of fermentation in plant tissues, and
both derive from the final product of glycolysis, i.e. pyruvate. The latter is converted to
acetaldehyde by the enzyme pyruvate decarboxylase (PDC). Subsequently, alcohol
dehydrogenase (ADH) catalyzes the conversion of acetaldehyde to ethanol, yielding NAD+
available for glycolysis. Lactate is formed via the activity of the enzyme lactate
dehydrogenase (LDH), which uses pyruvate as substrate, NAD+ being formed accordingly
(Bailey-Serres and Voesenek, 2008; Tadege et al., 1999). Lactate fermentation is often
related to cytosolic acidification, although the correlation between increase in acid lactic
and fall in pH is not always observed. Disruption in membrane-bound ATPase activity may
also be involved in acidosis of the cytosol. As ATP becomes limited under hypoxia, the
hydrolysis of ATP by ATPases is impaired, along with protons pumping across the
membrane, leading to acidification of the cytoplasm (Ferreira de Sousa and Sodek, 2002).
The activity of the enzyme ADH drastically increased in white clover under flooding stress.
Natural populations of white clover show significant differences in constitutive and induced
levels of ADH, with greater ADH activity correlated to improved flood tolerance (Chan and
Burton, 1992).
Adaptation to hypoxic stress often involves the gaseous plant hormone ethylene. The latter
accumulates in flooded soils, submerged plant organs, as well as in aerobic parts. The last
step in the synthesis of ethylene is O2-dependent and does not occur in anaerobic roots.
72
Hence, the immediate precursor of ethylene, i.e. 1-amino cyclopropane 1-carboxylic acid
(ACC), is transferred to the aerobic parts of the plants, where the reaction can take place.
Given its readily diffusible ability within the plant, the phytohormone may then accumulate
in anoxic roots (Sairam et al., 2008). Ethylene is involved in the formation of air-filled
tissues in the roots, called aerenchymas, which allow gas exchange from aerobic shoots to
anaerobic roots (Bailey-Serres and Voesenek, 2008; Sairam et al., 2008). This process is
well-established in flooding-tolerant species of Trifolium (Gibberd et al., 2001).
Aerenchymatous tissues may form constitutively, be induced in already mature tissues, or
may develop in newly formed adventitious roots. Two main types of aerenchymatous
tissues have been described. Lysigenous aerenchyma is formed through cell death events
with collapse of one or several cells, whereas schizogenous aerenchyma results from the
detachment of adjacent cells (Bailey-Serres and Voesenek, 2008; Wegner, 2010).
Aerotrophic rooting is an important trait for plants’ tolerance towards flooding, tolerant
species being more likely to have greater porosity than sensitive species (Gibberd et al.,
2001).
The production of ROS is a common outcome of many stress conditions, including
hypoxia. Under anaerobic conditions, as a consequence of a high intracellular redox status
and restricted energy availability, ROS are likely to be produced (Blokhina et al., 2003).
Anaerobic tissues may accumulate ROS via mitochondrial- and enzymatic-dependent
pathways, which may significantly damage plant cell’s components and jeopardize its
integrity (Sairam et al., 2008). The formation of activated oxygen, such as O2•- and H2O2,
may be facilitated through the activity of the enzyme xanthine oxidoreductase, which uses
acetaldehyde (formed as a by product of fermentation) and hypoxanthine (product of ATP
catabolism) as reductants (Blokhina and Fagerstedt, 2010). Furthermore, as reviewed by
Sairam et al. (2008), hypoxia induced the expression and the activity of NADPH oxidase in
pigeon pea plants, suggesting that the enzyme may be involved in ROS production under
O2 depletion. Oxidative damage has also been proposed to occur after re-aeration, a
phenomenon called “O2 paradox”, although the exact mechanisms involved are not yet
fully understood. In accordance with this, several studies showed that the production of
end-products of lipid peroxidation in hypoxic plant tissues was increased after re-aeration,
suggesting that oxidative damage was enhanced when O2 became suddenly available
73
(Rawyler et al., 2002). Hence, it would not be surprising for land plants that encounter
flooding conditions to display oxidative stress in submerged organs with enhanced
generation of ROS and mediation of ROS-scavengers (Simova-Stoilova et al., 2012).
Moreover, under O2-limited conditions, enhanced expression of enzymes operating in ROS
signalling and removal have been reported in several species, although little is known about
the mechanisms involved (Bailey-Serres and Voesenek, 2008). The ability to trigger
antioxidative mechanisms under O2 deprivation may strongly depend on the degree of
tolerance of the plant to that stress, several studies having reported contradictory results
with enhanced and decreased antioxidant levels (Blokhina et al., 2003). In barley, SOD
activity significantly increased in the roots and leaves under hypoxic conditions
(Kalashnikov et al., 1994), whereas decline in SOD activity in corn leaves was correlated to
O2•- accumulation under flooding stress (Yan et al., 1996). Another study of the effect of
soil flooding on barley plants showed enhanced activity of peroxidase, CAT and APX in
the leaves (Yordanova et al., 2004). Likewise, in waterlogged roots of eggplants and
tomatoes, APX activity significantly raised, total and reduced ascorbate/glutathione
moderately increased, whereas the activities of SOD, CAT, and GR were not affected (Lin
et al., 2004). Additionally, as a result of excess water in soil, products of anaerobic
metabolism by micro-organisms (such as Mn2+, Fe2+, hydrogen sulphide, acetic and butyric
acid) may accumulate to toxic levels and dramatically injure plant tissues (Drew, 1997;
Jackson and Colmer, 2005).
In contrast to other clover species, white clover is thought to be relatively tolerant to
flooding stress, one study showed flooded clovers with increased dry matter and nitrogen
fixation (Pugh et al., 1995). However, prolonged flooding treatment in both Trifolium
repens and Trifolium pratense induced decreases in chlorophyll a and b content, as well as
in carotenoids (Simova-Stoilova et al., 2012). The effect of flooding in white clover, from
different studies, seems to be slightly contradictory. Some authors reported flooded white
clover with significant leaf stress symptoms, growth inhibition and lateral root formation
(Simova-Stoilova et al., 2012). Others have shown reduction in the number of ramets and
total weight, but enhanced root porosity and secondary root development, and increased
leaf area and petiole length (Huber et al., 2009). In agreement with this, it is well accepted
that white clover displays high intra-specific variability towards flooding tolerance
74
(Jackson et al., 2009). The objectives of this chapter are to determine whether T. repens
Huia & Kopu II plants respond negatively to a root-flooding treatment by assessing the
effects of that stress on growth performance, oxidative damage and antioxidative defence
mechanisms. The assumption of whether the plant can or cannot cope with floodinginduced stress while infected with WClMV will be emphasized throughout the study.
75
Hypoxia
ATPase
dysfunction
Ethylene
NAD+
ADP
Glycolysis
ATP
NADH
Aerenchyma
formation
Cytosolic
acidification
Lactate
LHD
NAD+
Pyruvate
NADH
PDC
Xanthine
oxidoreductase
NADPH
oxidase
Mitochondrialdepend pathway
Acetaldehyde
NADH
ADH
NAD+
ROS
Ethanol
Figure 31 General overview of metabolic pathways and ROS generation under hypoxic conditions. ADH = alcohol dehydrogenase, ADP = adenosine
diphosphate, ATP = adenosine triphosphate, LHD = lactate dehydrogenase, NAD +/NADH = nicotinamide adenine dinucleotide (reduced/oxidized forms), PDC =
pyruvate decarboxylase, ROS = reactive oxygen species (Bailey-Serres and Voesenek, 2008; Blokhina et al., 2003; Ferreira de Sousa and Sodek, 2002; Sairam et
al., 2008; Tadege et al., 1999).
76
4.2 Methods
To investigate the effect of a root-flooding treatment on white clover’s performance and
oxidative damage, the same plants from Experiment 1 (i.e. previous Chapter) were used.
Following the final harvest of Experiment 1, the plants were allowed to recover for a period
of 4 weeks.
For both cultivars, non-infected and WClMV-infected plants were randomly selected and
assigned to a control and a root-flooding treatment. For Huia, 13 WClMV-/12 WClMV+
plants were used as controls, and 13 WClMV-/13 WClMV+ were root-flooded. For Kopu II,
12 WClMV-/12 WClMV+ plants were allocated to the control treatment, whereas 13
WClMV-/12 WClMV+ pots were subjected to flooding. The plants selected as root-flooded
were placed on a water-filled bench which flooded the pots to approximately two-thirds of
their height (Fig. 32), while the controls were assigned to another bench in similar
conditions used as in Experiment 1. It is worth mentioning that the plants were not fully
submerged and the upper part of the root system was in fact aerated. In this experiment, the
term “waterlogging” will then refer to “root-flooding” for which the bulk of the root
system, but not the whole, is considered submerged. An 8-day time-course experiment (day
0 to day 7) was carried out, for which 3 randomly-selected plants per cultivar and
treatment, i.e. WClMV-/WClMV+ vs root-flooded-/root-flooded+, were destructively
harvested on day 0, 2, 5 & 7. For day 0, only non-waterlogged plants were harvested as it
was unlikely that the treatment would have significantly affected the plants the same day it
was applied. In contrast to Experiment 1, both above- and below-ground parts of each plant
were collected.
77
Figure 32 Experimental design showing white clover Huia (yellow labels) and Kopu II (blue labels) placed
on a water-filled bench (M. Corral).
Measurements of chlorophyll fluorescence (PAM) were conducted every day on 3
randomly-selected replicates per treatment, for which Fv/Fm values were determined for 3
leaves and subsequently averaged (refer to section 2.1.4). At each harvest, shoot parts and
root samples were collected (only above-ground material was weighed) and subsequently
frozen in liquid nitrogen and stored at -80°C prior to biochemical analysis (see section 2.2).
Below is a summary of harvests and chlorophyll measurements undertaken over the time of
the experiment:
Harvest 1
PAM 1
PAM 2
Harvest 2
PAM 3
PAM 4
PAM 5
Harvest 3
PAM 6
PAM 7
Harvest 4
PAM 8
Day 0
Day 1
Day 2
Day 3
Day 4
Day 5
Day 6
Day 7
Figure 33 Timeline showing PAM measurements and harvests of Experiment 2.
78
4.3 Results
In the current experiment, both uninfected and WClMV-infected plants that were not
waterlogged are referred to as “controls” throughout the following section. Also, in order to
make the figures more explicit and clear, data related to root-flooded plants was labelled
“Uninfected + Rf” and “WClMV + Rf” (Rf = root-flooded).
4.3.1
Effect of root-flooding on the root system
Waterlogging treatment had a strong impact on root development. Figure 34 shows the root
systems of a control and a flooded plant approximately 4 weeks following the experiment,
as the plants were still kept under excess water conditions. As highlighted, drained controls
displayed healthy and dense root systems (Fig. 34a) with no indication of stress, while
flooded roots (Fig. 34b) appeared brownish (red arrow), a response most likely due to a
build up of phenolics, with a noticeable proportion of roots having died. Flooded plants
also grew roots near the soil surface (yellow arrow), where oxygen was available. Although
no further biochemical analyses were performed at that stage (i.e. post-experiment), it
seemed worthy of mention that none of the plants subjected to root-flooding had perished.
a
Control plant
b
Root-flooded plant
Figure 34 Comparison of root systems between a control and a root-flooded plant (M. Corral). Red arrows =
necrotic roots / Yellow arrows = roots formed near the surface.
79
Furthermore, even though roots appeared to negatively respond to submergence, a high
proportion of flooded plants developed roots in the water, either emerging from the bottom
of the pots or grown from newly formed stolons. Interestingly, in contrast to controls, roots
growing under water did not indicate any sign of nodulation (Fig. 36). Studies of crosssections of submerged roots showed enhanced formation of programme cell death-induced
lysigenous aerenchymas (Fig. 35, red arrows).
a
b
100 µm
Figure 35 Cross-sections of non-waterlogged (a) and waterlogged (b) roots stained with toluidine blue. Red
arrows indicate root lysigenous aerenchymas (M. Corral). Root samples were collected from controls and
root-flooded plants 5 weeks post-treatment.
80
a
b
c
0.5 cm
Figure 36 Root-flooded plants showing root formation in the water (5 weeks post-waterlogging); (a) roots emerging from the bottom of the pot, (b) roots formed
on newly developed stolons, and (c) typical white clover nodules (M. Corral).
81
4.3.2
Effect of WClMV & root-flooding on growth parameters
4.3.2.1 Above-ground fresh weight
Huia
Uninfected
WClMV
Uninfected + Rf
WClMV + Rf
40
Mean Fresh Weight (g per plant)
R² = 0.998
30
R² = 0.6916
20
R² = 0.9334
10
R² = 0.3787
0
0
1
2
3
4
5
6
7
Time-course (Day)
Figure 37 Above-ground fresh weight of uninfected (♦), WClMV-infected (♦), uninfected/root-flooded (●)
and WClMV-infected/root-flooded (●) white clover Huia throughout the time-course experiment (n = 3).
Each line is a polynomial regression fit (2 nd degree) of the data of each treatment, with associated R² values.
Data for “Uninfected” and “WClMV” at day 0 were used as reference points for “Uninfected + Rf” and
“WClMV + Rf” at the corresponding day.
82
Kopu II
Uninfected
WClMV
Uninfected + Rf
WClMV + Rf
Mean Fresh Weight (g per plant)
20
R² = 0.7023
15
R² = 0.961
R² = 0.9992
10
R² = 0.9155
5
0
0
1
2
3
4
5
6
7
Time-course (Day)
Figure 38 Above-ground fresh weight of uninfected (♦), WClMV-infected (♦), uninfected/root-flooded (●)
and WClMV-infected/root-flooded (●) white clover Kopu II throughout the time-course experiment (n = 3).
Each line is a polynomial regression fit (2 nd degree) of the data of each treatment, with associated R² values.
Data from “Uninfected” and “WClMV” at day 0 were used as reference points for “Uninfected + Rf” and
“WClMV + Rf” at the corresponding day.
Fresh weight (FW) was affected significantly by treatment and time in both cultivars (Huia,
P < 0.001; Kopu II, P < 0.05 for both factors). In Huia, uninfected and WClMV-treated
plants that were not root-flooded showed increases in FW, being greater in uninfected
plants. FW in uninfected-waterlogged plants augmented in the first few days and
subsequently reached a steady state, while WClMV-waterlogged subjects displayed a
constant decline throughout the experiment (Fig. 37). Growth patterns in Kopu II were
different. Both uninfected controls and Rf-uninfected plants accumulated biomass, whereas
infected ones did not show any increase (Fig. 38).
83
4.3.2.2 Dry matter content
Uninfected
Uninfected + Rf
Mean Dry Matter Content
(g per plant)
5
4.5
WClMV
WClMV + Rf
a
4
3.5
3
ab
ab
a
2.5
a
2
b
1.5
b
1
b
0.5
0
Huia
Kopu II
Figure 39 Dry matter content of above-ground biomass of uninfected (□), WClMV-infected (■),
uninfected/root-flooded (■) and WClMV-infected/root-flooded (■) white clover Huia (left) and Kopu II
(right). Error bars indicate the standard error of the mean for each treatment. Groups were compared using
generalized linear models (GLM) with Bonferroni post-hoc analysis. Bars with different letters are
significantly different.
In both cultivars, dry matter content (DMC) was affected significantly by treatment (P <
0.001 & P < 0.01 for Huia and Kopu II, respectively). A time effect was only found in Huia
(P < 0.001). In Huia, DMC was significantly reduced only in infected plants subjected to
waterlogging, showing a 67% decrease when compared to uninfected controls. In contrast,
both well drained and submerged WClMV-infected Kopu II had reduced DMC, being
~55% lower than in controls (Fig. 39).
84
4.3.2.3 Photosynthetic efficiency
Photosynthetic efficiency
(Fv/Fm)
0.84
Uninfected
WClMV
Uninfected + Rf
WClMV + Rf
0.80
0.76
0.72
0.68
0.64
0.60
Huia
Kopu II
Figure 40 Maximal photochemical efficiency (Fv/Fm) of uninfected (□), WClMV-infected (■),
uninfected/root-flooded (■) and WClMV-infected/root-flooded (■) white clover Huia (left) and Kopu II
(right) during the time-course experiment (data given as mean value). Error bars indicate the standard error of
the mean for each treatment.
Maximal photochemical efficiency was not affected by treatment. A time effect was
suspected in both Huia (P < 0.05) and Kopu II (P < 0.01), however t-tests did not reveal
any significant differences (mean Fv/Fm values ranging from 0.739 to 0.798, SD ± 0.03)
(Fig. 40).
85
4.3.3
Markers of oxidative stress
4.3.3.1 Protein carbonyls
SHOOT
Huia
c
20
10
b
a a ab
a a
a a
a a
b
0
a a
10
b
20
ROOT
Protein carbonyls
(nmol.mg-1 protein)
30
c
b
b
30
b
40
50
c
Day 0
Day 2
Day 5
WClMV + Rf
Uninfected + Rf
WClMV
Uninfected
WClMV + Rf
Uninfected + Rf
WClMV
Uninfected
WClMV + Rf
Uninfected + Rf
WClMV
Uninfected
WClMV + Rf
Uninfected + Rf
WClMV
Uninfected
60
Day 7
Figure 41 Protein carbonyl content in uninfected (□), WClMV-infected (■), uninfected/root-flooded (■) and
WClMV-infected/root-flooded (■) Huia plants at each harvest (day 0, 2, 5 & 7). Error bars indicate the
standard error of the mean for each treatment. Groups were compared using generalized linear models (GLM)
with Bonferroni post-hoc analysis. Within each time point and organ, bars with different letters are
significantly different.
86
SHOOT
c
30
20
10
b
a a ab
a a
a a
a a
b
0
a a
10
20
b
c
30
ROOT
Protein carbonyls
(nmol.mg-1 protein)
Kopu II
b
40
b
b
50
60
c
Day 0
Day 2
Day 5
Uninfected
WClMV
Uninfected + Rf
WClMV + Rf
Uninfected
WClMV
Uninfected + Rf
WClMV + Rf
Uninfected
WClMV
Uninfected + Rf
WClMV + Rf
Uninfected
WClMV
Uninfected + Rf
WClMV + Rf
70
Day 7
Figure 42 Protein carbonyl content in uninfected (□), WClMV-infected (■), uninfected/root-flooded (■) and
WClMV-infected/root-flooded (■) Kopu II plants at each harvest (day 0, 2, 5 & 7). Error bars indicate the
standard error of the mean for each treatment. Groups were compared using generalized linear models (GLM)
with Bonferroni post-hoc analysis. Within each time point and organ, bars with different letters are
significantly different.
In both cultivars, protein carbonyl content was affected significantly by treatment, time and
organ (P < 0.001). Non-flooded plants (controls) did not show any variations in protein
carbonyls in shoots and roots, although shoots had on average higher levels than roots (P <
0.01). However, root-flooded plants showed a gradual and significant accumulation of
protein carbonyls in both organs, an earlier increase (day 2) and higher contents being
found in roots (P < 0.05). At the end of the experiment, in both cultivars, protein carbonyls
increased by at least 2-fold and 4-fold in shoots of uninfected and WClMV-infected
flooded plants, respectively. In roots, it increased by at least 5-fold in uninfected plants and
10-fold in virus-treated plants prone to root-flooding (Figs 41 & 42).
87
4.3.3.2 Lipid peroxides
60
c
40
20
b
a a ab
a a
a a
a a
b
0
a a
20
b
40
ROOT
Lipid Peroxides
(nmol.g-1 FW)
SHOOT
Huia
c
b
60
b
b
80
100
c
Day 0
Day 2
Day 5
Uninfected
WClMV
Uninfected + Rf
WClMV + Rf
Uninfected
WClMV
Uninfected + Rf
WClMV + Rf
Uninfected
WClMV
Uninfected + Rf
WClMV + Rf
Uninfected
WClMV
Uninfected + Rf
WClMV + Rf
120
Day 7
Figure 43 Lipid peroxide content in uninfected (□), WClMV-infected (■), uninfected/root-flooded (■) and
WClMV-infected/root-flooded (■) Huia plants at each harvest (day 0, 2, 5 & 7). Error bars indicate the
standard error of the mean for each treatment. Groups were compared using generalized linear models (GLM)
with Bonferroni post-hoc analysis. Within each time point and organ, bars with different letters are
significantly different.
88
80
60
40
20
0
20
40
60
80
100
120
140
c
a a
b
b
b
a a ab
a a
a a
a a
b
b
b
b
Day 0
Day 2
Day 5
Uninfected
WClMV
Uninfected + Rf
WClMV + Rf
Uninfected
WClMV
Uninfected + Rf
WClMV + Rf
Uninfected
WClMV
Uninfected + Rf
WClMV + Rf
c
Uninfected
WClMV
Uninfected + Rf
WClMV + Rf
SHOOT
ROOT
Lipid Peroxides
(nmol.g-1 FW)
Kopu II
Day 7
Figure 44 Lipid peroxide content in uninfected (□), WClMV-infected (■), uninfected/root-flooded (■) and
WClMV-infected/root-flooded (■) Kopu II plants at each harvest (day 0, 2, 5 & 7). Error bars indicate the
standard error of the mean for each treatment. Groups were compared using generalized linear models (GLM)
with Bonferroni post-hoc analysis. Within each time point and organ, bars with different letters are
significantly different.
Lipid peroxide (LP) content was affected significantly by treatment, time and organ in each
cultivar (P < 0.001). Controls did not display any differences, except that levels of lipid
peroxidation were higher in shoots than roots for both WClMV- and WClMV+ plants (P <
0.05). In contrast, LP significantly increased in root-flooded plants with higher activity in
roots (P < 0.05) in both virus treatments and cultivars. At day 7, in both cultivars, levels of
lipid peroxidation increased by up to 80% and at least 225% in shoots of uninfected and
infected flooded plants, respectively. In flooded roots, it increased by at least 4-fold in
WClMV- and 9-fold in WClMV+ plants. Rf-infected plants had higher lipid peroxide
contents in both organs as compared to virus-free plants (P < 0.001) (Figs 43 & 44).
89
4.3.4
Non-enzymatic antioxidants
4.3.4.1 Total ascorbate levels
Huia
b
SHOOT
100
a ab
50
b ab
c
b b
a a
a a
a a
a a
0
a a
50
b b
100
b b
c
b
Day 0
Day 2
Uninfected
WClMV
Uninfected + Rf
WClMV + Rf
Uninfected
WClMV
Uninfected + Rf
WClMV + Rf
Uninfected
WClMV
Uninfected + Rf
WClMV + Rf
150
Day 5
Uninfected
WClMV
Uninfected + Rf
WClMV + Rf
ROOT
Total Ascorbate
(µmol.g-1 FW)
150
Day 7
Figure 45 Total ascorbate concentration in uninfected (□), WClMV-infected (■), uninfected/root-flooded (■)
and WClMV-infected/root-flooded (■) Huia plants at each harvest (day 0, 2, 5 & 7). Error bars indicate the
standard error of the mean for each treatment. Groups were compared using generalized linear models (GLM)
with Bonferroni post-hoc analysis. Within each time point and organ, bars with different letters are
significantly different.
90
b
200
150
100
a ab
50
b ab
c
b b
a a
a a
a a
a a
0
a a
50
b b
100
b b
c
150
b
Day 0
Day 2
Uninfected
WClMV
Uninfected + Rf
WClMV + Rf
Uninfected
WClMV
Uninfected + Rf
WClMV + Rf
Uninfected
WClMV
Uninfected + Rf
WClMV + Rf
200
Day 5
Uninfected
WClMV
Uninfected + Rf
WClMV + Rf
ROOT
Total Ascorbate
(µmol.g-1 FW)
SHOOT
Kopu II
Day 7
Figure 46 Total ascorbate concentration in uninfected (□), WClMV-infected (■), uninfected/root-flooded (■)
and WClMV-infected/root-flooded (■) Kopu II plants at each harvest (day 0, 2, 5 & 7). Error bars indicate the
standard error of the mean for each treatment. Groups were compared using generalized linear models (GLM)
with Bonferroni post-hoc analysis. Within each time point and organ, bars with different letters are
significantly different.
In both cultivars, total ascorbate was affected significantly by treatment, time and organ (P
< 0.001). Non-flooded plants did not show any differences in both shoots/roots, although
roots had lower amount of ascorbate than shoots (P < 0.01). However, waterlogged plants
showed a constant and significant increase in both organs (from day 2), with no differences
found between organs. At day 7, both cultivars displayed increased ascorbate by up to 3fold and 2-fold in shoots of uninfected and virus-treated waterlogged plants, respectively.
In flooded roots, ascorbate increased by at least 3-fold in uninfected plants and up to 2-fold
in infected ones. Overall, levels of ascorbate were lower in waterlogged/WClMV+ as
compared to waterlogged/WClMV- in both cultivars and organs (P < 0.01) (Figs 45 & 46).
91
4.3.4.2 Reduced ascorbate levels
SHOOT
100
a a
a a
ab a
75
c bc
b b
b b
b b
b b
b b
50
25
0
ROOT
% Reduced Ascorbate
Huia
25
50
75
a a
100
a a
a a
Day 0
Day 2
Day 5
WClMV + Rf
Uninfected + Rf
WClMV
Uninfected
WClMV + Rf
Uninfected + Rf
WClMV
Uninfected
WClMV + Rf
Uninfected + Rf
WClMV
Uninfected
WClMV + Rf
Uninfected + Rf
WClMV
Uninfected
125
Day 7
Figure 47 % Reduced ascorbate in uninfected (□), WClMV-infected (■), uninfected/root-flooded (■) and
WClMV-infected/root-flooded (■) Huia plants at each harvest (day 0, 2, 5 & 7). Error bars indicate the
standard error of the mean for each treatment. Groups were compared using generalized linear models (GLM)
with Bonferroni post-hoc analysis. Within each time point and organ, bars with different letters are
significantly different.
92
SHOOT
a a
a a
100
75
a a
b b
b b
b b
b b
b b
b b
50
25
0
ROOT
% Reduced Ascorbate
Kopu II
25
50
75
a a
a a
a a
100
Day 0
Day 2
Day 5
WClMV + Rf
Uninfected + Rf
WClMV
Uninfected
WClMV + Rf
Uninfected + Rf
WClMV
Uninfected
WClMV + Rf
Uninfected + Rf
WClMV
Uninfected
WClMV + Rf
Uninfected + Rf
WClMV
Uninfected
125
Day 7
Figure 48 % Reduced ascorbate in uninfected (□), WClMV-infected (■), uninfected/root-flooded (■) and
WClMV-infected/root-flooded (■) Kopu II plants at each harvest (day 0, 2, 5 & 7). Error bars indicate the
standard error of the mean for each treatment. Groups were compared using generalized linear models (GLM)
with Bonferroni post-hoc analysis. Within each time point and organ, bars with different letters are
significantly different.
Reduced ascorbate was affected by treatment and time (P < 0.001) in both cultivars, and by
organ only in Huia (P < 0.05). The proportion of reduced ascorbate in non-flooded plants
did not differ through the experiment. In contrast, it significantly decreased in both
uninfected and WClMV-treated plants prone to root-flooding, in both shoots and roots from
day 2. No major differences were found between shoots and roots, except for Huia, in
which uninfected/non-waterlogged roots had slightly higher content of reduced ascorbate
(P < 0.05). In the two cultivars, at the end of the experiment, the proportion of reduced
ascorbate decreased by approximately 25% in shoots and roots of uninfected and infected
root-flooded plants (Figs. 47 & 48).
93
4.3.4.3 Total glutathione levels
SHOOT
b
300
200
ab a b b
100
c
a a
0
100
a
200
300
a
c
400
500
b
Day 0
Day 2
Uninfected
WClMV
Uninfected + Rf
WClMV + Rf
Uninfected
WClMV
Uninfected + Rf
WClMV + Rf
Uninfected
WClMV
Uninfected + Rf
WClMV + Rf
600
Day 5
Uninfected
WClMV
Uninfected + Rf
WClMV + Rf
ROOT
Total Glutathione
(nmol.g-1 FW)
Huia
Day 7
Figure 49 Total glutathione concentration in uninfected (□), WClMV-infected (■), uninfected/root-flooded
(■) and WClMV-infected/root-flooded (■) Huia plants at each harvest (day 0, 2, 5 & 7). Error bars indicate
the standard error of the mean for each treatment. Groups were compared using generalized linear models
(GLM) with Bonferroni post-hoc analysis. Within each time point and organ, bars with different letters are
significantly different.
94
b
400
300
200
100
0
100
200
300
400
500
600
700
ab a
b b
c
a a
a
a
c
Day 0
Day 2
Day 5
Uninfected
WClMV
Uninfected + Rf
WClMV + Rf
Uninfected
WClMV
Uninfected + Rf
WClMV + Rf
Uninfected
WClMV
Uninfected + Rf
WClMV + Rf
b
Uninfected
WClMV
Uninfected + Rf
WClMV + Rf
SHOOT
ROOT
Total Glutathione
(nmol.g-1 FW)
Kopu II
Day 7
Figure 50 Total glutathione concentration in uninfected (□), WClMV-infected (■), uninfected/root-flooded
(■) and WClMV-infected/root-flooded (■) Kopu II plants at each harvest (day 0, 2, 5 & 7). Error bars indicate
the standard error of the mean for each treatment. Groups were compared using generalized linear models
(GLM) with Bonferroni post-hoc analysis. Within each time point and organ, bars with different letters are
significantly different.
In both cultivars, treatment, time and organ had an effect on total glutathione (P < 0.001).
Levels of glutathione did not differ in shoots and roots of controls, although they were
significantly higher in roots than in shoots (P < 0.001). In flooded plants, glutathione
started to rise at day 5 in shoots and day 7 in roots. At day 7, glutathione levels in Huia
increased by up to 180% in shoots and roots of Rf-uninfected plants, and about 70% in both
organs of Rf-WClMV+ plants. In Kopu II, it increased by about 280% in shoots and 140%
in roots of waterlogged/uninfected plants, and by 100% in shoots and 40% in roots of
waterlogged/WClMV+ plants. Root-flooded/uninfected plants had greater total glutathione
levels than root-flooded/infected plants (P < 0.001) (Figs 49 & 50).
95
4.3.4.4 Reduced glutathione levels
SHOOT
a a
a a
a a
100
75
b b
b b
b b
b b
b b
b b
50
25
0
ROOT
% Reduced Glutathione
Huia
25
50
75
a a
100
a a
a a
Day 0
Day 2
Day 5
WClMV + Rf
Uninfected + Rf
WClMV
Uninfected
WClMV + Rf
Uninfected + Rf
WClMV
Uninfected
WClMV + Rf
Uninfected + Rf
WClMV
Uninfected
WClMV + Rf
Uninfected + Rf
WClMV
Uninfected
125
Day 7
Figure 51 % Reduced glutathione in uninfected (□), WClMV-infected (■), uninfected/root-flooded (■) and
WClMV-infected/root-flooded (■) Huia plants at each harvest (day 0, 2, 5 & 7). Error bars indicate the
standard error of the mean for each condition. Groups were compared using generalized linear models (GLM)
with Bonferroni post-hoc analysis. Within each time point and organ, bars with different letters are
significantly different.
96
SHOOT
a a
a a
a a
100
75
50
b b
b b
b b
b b
b b
b b
25
0
ROOT
% Reduced Glutathione
Kopu II
25
50
75
100
a a
a a
a a
Day 0
Day 2
Day 5
WClMV + Rf
Uninfected + Rf
WClMV
Uninfected
WClMV + Rf
Uninfected + Rf
WClMV
Uninfected
WClMV + Rf
Uninfected + Rf
WClMV
Uninfected
WClMV + Rf
Uninfected + Rf
WClMV
Uninfected
125
Day 7
Figure 52 % Reduced glutathione in uninfected (□), WClMV-infected (■), uninfected/root-flooded (■) and
WClMV-infected/root-flooded (■) Kopu II plants at each harvest (day 0, 2, 5 & 7). Error bars indicate the
standard error of the mean for each treatment. Groups were compared using generalized linear models (GLM)
with Bonferroni post-hoc analysis. Within each time point and organ, bars with different letters are
significantly different.
In both cultivars, reduced glutathione was affected significantly by treatment and time (P <
0.001), no differences being found between organs. Levels of reduced glutathione did not
differ in non-waterlogged controls (uninfected and WClMV-infected). However, it
significantly decreased in virus-free and infected waterlogged plants, from day 2, in both
shoots and roots (P < 0.001 in each condition). At day 7, in both cultivars/organs, the
proportion of reduced glutathione dropped by up to 40% in uninfected and virus-treated
plants prone to root-flooding (Figs 51 & 52).
97
4.3.5
Enzymatic antioxidants
SHOOT
Huia
300
b
200
ab a
100
c
b ab
a a
0
a a
a a
100
a a
b b
b b
200
c
300
b
Day 0
Day 2
Day 5
WClMV + Rf
Uninfected + Rf
WClMV
Uninfected
WClMV + Rf
Uninfected + Rf
WClMV
Uninfected
WClMV + Rf
Uninfected + Rf
WClMV
Uninfected
WClMV + Rf
Uninfected + Rf
WClMV
400
Uninfected
ROOT
SOD Activity (units. min-1.mg-1 protein)
4.3.5.1 Superoxide dismutase
Day 7
Figure 53 Superoxide dismutase activity in uninfected (□), WClMV-infected (■), uninfected/root-flooded (■)
and WClMV-infected/root-flooded (■) Huia plants at each harvest (day 0, 2, 5 & 7). Error bars indicate the
standard error of the mean for each treatment. Groups were compared using generalized linear models (GLM)
with Bonferroni post-hoc analysis. Within each time point and organ, bars with different letters are
significantly different.
98
SHOOT
400
b
300
200
c
b ab
a a
100
a a
0
100
a
a a
a a
a
200
b b
b b
c
300
b
Day 0
Day 2
Day 5
WClMV + Rf
Uninfected + Rf
WClMV
Uninfected
WClMV + Rf
Uninfected + Rf
WClMV
Uninfected
WClMV + Rf
Uninfected + Rf
WClMV
Uninfected
WClMV + Rf
Uninfected + Rf
WClMV
400
Uninfected
ROOT
SOD Activity (units.min-1.mg-1 protein)
Kopu II
Day 7
Figure 54 Superoxide dismutase activity in uninfected (□), WClMV-infected (■), uninfected/root-flooded (■)
and WClMV-infected/root-flooded (■) Kopu II plants at each harvest (day 0, 2, 5 & 7). Error bars indicate the
standard error of the mean for each treatment. Groups were compared using generalized linear models (GLM)
with Bonferroni post-hoc analysis. Within each time point and organ, bars with different letters are
significantly different.
In both cultivars, SOD activity was affected significantly by treatment, time (P < 0.001)
and organ (P < 0.01). Controls revealed no changes in SOD in both organs, although roots
had lower enzymatic activity than in shoots (P < 0.05). In waterlogged subjects, SOD
increased from day 2 in both organs, although no differences were found at day 5 in shoots.
At day 7, in Huia, SOD activity increased by 220% in shoots and 350% in roots of Rfuninfected plants, and by 67% in shoots and 180% in roots of Rf-WClMV+ plants. In Kopu
II, it increased by about 200% in both shoots/roots of Rf-uninfected plants, and by 126% in
shoots and 87% in roots of Rf-infected plants. Overall, flooded/virus-free plants displayed
higher SOD activity than flooded/infected plants (P < 0.001) (Figs 53 & 54).
99
4.3.5.2 Ascorbate peroxidase
Huia
b
SHOOT
b
2.0
1.5
b
ab
1.0
a a
a a
a a
a a
0.5
0.5
ROOT
APX Activity
(µmol. min-1.mg-1 protein)
2.5
a a
1.0
b b
1.5
c
b
c
b
Day 0
Day 2
Day 5
Uninfected
WClMV
Uninfected + Rf
WClMV + Rf
Uninfected
WClMV
Uninfected + Rf
WClMV + Rf
Uninfected
WClMV
Uninfected + Rf
WClMV + Rf
Uninfected
WClMV
Uninfected + Rf
WClMV + Rf
2.0
Day 7
Figure 55 Ascorbate peroxidase activity in uninfected (□), WClMV-infected (■), uninfected/root-flooded (■)
and WClMV-infected/root-flooded (■) Huia plants at each harvest (day 0, 2, 5 & 7). Error bars indicate the
standard error of the mean for each treatment. Groups were compared using generalized linear models (GLM)
with Bonferroni post-hoc analysis. Within each time point and organ, bars with different letters are
significantly different.
100
Kopu II
SHOOT
b
2.5
2.0
b
1.5
c
ab
a a
1.0
a a
0.5
0.5
ab ab
1.0
ac
a a
a
b
1.5
b
Day 0
Day 2
Uninfected
WClMV
Uninfected + Rf
WClMV + Rf
Uninfected
WClMV
Uninfected + Rf
WClMV + Rf
Uninfected
WClMV
Uninfected + Rf
WClMV + Rf
2.0
Day 5
Uninfected
WClMV
Uninfected + Rf
WClMV + Rf
ROOT
APX Activity
(µmol. min-1.mg-1 protein)
3.0
Day 7
Figure 56 Ascorbate peroxidase activity in uninfected (□), WClMV-infected (■), uninfected/root-flooded (■)
and WClMV-infected/root-flooded (■) Kopu II plants at each harvest (day 0, 2, 5 & 7). Error bars indicate the
standard error of the mean for each treatment. Groups were compared using generalized linear models (GLM)
with Bonferroni post-hoc analysis. Within each time point and organ, bars with different letters are
significantly different.
Activity of APX was significantly affected by treatment, time and organ (P < 0.001) in the
two cultivars. Non-waterlogged uninfected and WClMV-treated plants did not show any
differences in APX activity, except that it was lower in roots than in shoots (P < 0.01).
Root-flooded plants had increased levels of APX, with, overall, greater activity observed in
Rf-uninfected plants. At the end of the experiment, in Huia, APX activity increased by
230% in shoots and roots of waterlogged/virus-free plants, and by up to 130% in shoots and
roots of waterlogged/infected ones. In Kopu II, it increased by 290% in shoots and 80% in
roots of waterlogged/uninfected plants, and by about 70% in shoots and 40% in roots of
waterlogged/infected plants (Figs 55 & 56).
101
4.3.5.3 Glutathione peroxidase
Huia
b
SHOOT
ab b
300
b
a a
a ac
a a
a a
c
c
a a
100
100
ROOT
GPX Activity
(nmol. min-1.mg-1 protein)
500
300
a a
b b
b
500
c
c
b
Day 0
Day 2
Day 5
Uninfected
WClMV
Uninfected + Rf
WClMV + Rf
Uninfected
WClMV
Uninfected + Rf
WClMV + Rf
Uninfected
WClMV
Uninfected + Rf
WClMV + Rf
Uninfected
WClMV
Uninfected + Rf
WClMV + Rf
700
Day 7
Figure 57 Glutathione peroxidase activity in uninfected (□), WClMV-infected (■), uninfected/root-flooded
(■) and WClMV-infected/root-flooded (■) Huia plants at each harvest (day 0, 2, 5 & 7). Error bars indicate
the standard error of the mean for each treatment. Groups were compared using generalized linear models
(GLM) with Bonferroni post-hoc analysis. Within each time point and organ, bars with different letters are
significantly different.
102
SHOOT
b
600
400
ab a
ab b
200
b
c
c
a ac
a a
a a
a a
0
a a
200
400
c
b b
c
b
600
b
Day 0
Day 2
Uninfected
WClMV
Uninfected + Rf
WClMV + Rf
Uninfected
WClMV
Uninfected + Rf
WClMV + Rf
Uninfected
WClMV
Uninfected + Rf
WClMV + Rf
800
Day 5
Uninfected
WClMV
Uninfected + Rf
WClMV + Rf
ROOT
GPX Activity
(nmol. min-1.mg-1 protein)
Kopu II
Day 7
Figure 58 Glutathione peroxidase activity in uninfected (□), WClMV-infected (■), uninfected/root-flooded
(■) and WClMV-infected/root-flooded (■) Kopu II plants at each harvest (day 0, 2, 5 & 7). Error bars indicate
the standard error of the mean for each treatment. Groups were compared using generalized linear models
(GLM) with Bonferroni post-hoc analysis. Within each time point and organ, bars with different letters are
significantly different.
In both cultivars, GPX activity was affected significantly by treatment and time (P <
0.001). An organ effect was found only in Huia (P < 0.001). Controls did not show any
variations in GPX levels, although higher activity was found in shoots (P < 0.001;
including Kopu II, despite the results from ANOVA). Root-flooded subjects had marked
increases in GPX when non-infected, being significantly higher than in infected plants (P <
0.001) at day 7. In Huia, after a week of root-flooding, levels of GPX enhanced by 160% in
shoots and 335% in roots of Rf-WClMV- plants, and by 40% in shoots and 216% in roots
of Rf-WClMV+ plants. In Kopu II, increases in GPX were about 200% in shoots and 240%
in roots of Rf-virus-free individuals, and about 100% in shoots and 130% in roots of Rfinfected plants (Figs. 57 & 58).
103
4.3.5.4 Catalase
SHOOT
b
300
c
b b
200
100
a a
a a
a a
a a
0
a a
100
b b
200
c
b b
300
b
Day 0
Day 2
Uninfected
WClMV
Uninfected + Rf
WClMV + Rf
Uninfected
WClMV
Uninfected + Rf
WClMV + Rf
Uninfected
WClMV
Uninfected + Rf
WClMV + Rf
400
Day 5
Uninfected
WClMV
Uninfected + Rf
WClMV + Rf
ROOT
CAT Activity
(µmol. min-1.mg-1 protein)
Huia
Day 7
Figure 59 Catalase activity in uninfected (□), WClMV-infected (■), uninfected/root-flooded (■) and
WClMV-infected/root-flooded (■) Huia plants at each harvest (day 0, 2, 5 & 7). Error bars indicate the
standard error of the mean for each treatment. Groups were compared using generalized linear models (GLM)
with Bonferroni post-hoc analysis. Within each time point and organ, bars with different letters are
significantly different.
104
Kopu II
SHOOT
b
300
c
b b
200
a a
a a
a a
a a
100
0
100
a a
200
ROOT
CAT Activity
(µmol. min-1.mg-1 protein)
400
b b
300
c
b b
400
b
Day 0
Day 2
Day 5
Uninfected
WClMV
Uninfected + Rf
WClMV + Rf
Uninfected
WClMV
Uninfected + Rf
WClMV + Rf
Uninfected
WClMV
Uninfected + Rf
WClMV + Rf
Uninfected
WClMV
Uninfected + Rf
WClMV + Rf
500
Day 7
Figure 60 Catalase activity in uninfected (□), WClMV-infected (■), uninfected/root-flooded (■) and
WClMV-infected/root-flooded (■) Kopu II plants at each harvest (day 0, 2, 5 & 7). Error bars indicate the
standard error of the mean for each treatment. Groups were compared using generalized linear models (GLM)
with Bonferroni post-hoc analysis. Within each time point and organ, bars with different letters are
significantly different.
CAT activity was affected by treatment and time (P < 0.001) in both cultivars, with an
organ effect detected only in Huia (P < 0.01). Non-flooded plants had no variations in
CAT, though greater activity was displayed in shoots (P < 0.01; including Kopu II, despite
the results from ANOVA). In flooded plants, levels of CAT increased in both shoots and
roots, with an earlier response in roots (day 2). At day 7, in Huia, CAT increased by 170%
in shoots and 350% in roots of Rf-uninfected plants, and by 77% in shoots and 275% in
roots of Rf-infected plants. In Kopu II, it increased by 170% in shoots and 270% in roots of
Rf-uninfected plants, and by 150% in shoots and 130% in roots of Rf-infected plants. All
together, these results show that Rf-WClMV- clovers had higher CAT activity than in RfWClMV+ plants, in both organs (P < 0.001) (Figs 59 & 60).
105
4.3.5.5 Glutathione S-transferase
In both cultivars, GST activities in shoots and roots were not affected by treatment or time
(P > 0.05). The only significant difference was found between organs, with roots having
greater GST levels in each treatment condition (P < 0.001).
4.3.5.6 Glutathione reductase
Huia
SHOOT
b
250
c
c bc
150
a ab
a a
a a
a a
50
50
a a
150
b b
c
b b
250
b
Day 0
Day 2
Uninfected
WClMV
Uninfected + Rf
WClMV + Rf
Uninfected
WClMV
Uninfected + Rf
WClMV + Rf
Uninfected
WClMV
Uninfected + Rf
WClMV + Rf
350
Day 5
Uninfected
WClMV
Uninfected + Rf
WClMV + Rf
ROOT
GR Activity
(nmol. min-1.mg-1 protein)
350
Day 7
Figure 61 Glutathione reductase activity in uninfected (□), WClMV-infected (■), uninfected/root-flooded (■)
and WClMV-infected/root-flooded (■) Huia plants at each harvest (day 0, 2, 5 & 7). Error bars indicate the
standard error of the mean for each treatment. Groups were compared using generalized linear models (GLM)
with Bonferroni post-hoc analysis. Within each time point and organ, bars with different letters are
significantly different.
106
Kopu II
SHOOT
b
300
c
c bc
200
100
a ab
a a
a a
a a
0
a a
100
b b
200
c
b b
300
b
Day 0
Day 2
Uninfected
WClMV
Uninfected + Rf
WClMV + Rf
Uninfected
WClMV
Uninfected + Rf
WClMV + Rf
Uninfected
WClMV
Uninfected + Rf
WClMV + Rf
400
Day 5
Uninfected
WClMV
Uninfected + Rf
WClMV + Rf
ROOT
GR Activity
(nmol. min-1.mg-1 protein)
400
Day 7
Figure 62 Glutathione reductase activity in uninfected (□), WClMV-infected (■), uninfected/root-flooded (■)
and WClMV-infected/root-flooded (■) Kopu II plants at each harvest (day 0, 2, 5 & 7). Error bars indicate the
standard error of the mean for each treatment. Groups were compared using generalized linear models (GLM)
with Bonferroni post-hoc analysis. Within each time point and organ, bars with different letters are
significantly different.
GR activity was affected significantly by treatment, time and organ (P < 0.001) in the two
cultivars. Non-waterlogged controls did not display any changes in GR levels throughout
the experiment, in both cultivars and organs, although greater activity was found in shoots
(P < 0.001). Increases in GR activity were observed in flooded plants from day 2 in roots
and day 5 in shoots, being more intense in uninfected subjects. At day 7, in Huia, GR
activity enhanced by 220% in shoots and 320% in roots of flooded/uninfected plants, and
by 65% in shoots and 135% in roots of flooded/infected ones. In Kopu II, it increased by
about 270% in shoots and roots of flooded/virus-free plants, and by 100% in shoots and
roots of flooded/virus-treated plants. In both cultivars, levels of GR were higher in
flooded/uninfected plants as compared to flooded/infected ones (P < 0.001) (Figs 61 & 62).
107
Table 9 Summary of ANOVA results for growth parameters, oxidative damage and antioxidant activities measured in Experiment 2
Cv. Huia
Treatmenta
Cv. Kopu II
Timeb
Organc
Treatmenta
Timeb
Organc
Growth parameters
Above-ground fresh weight
F3, 32 = 9.72, P < 0.001
F3, 32 = 8.60, P < 0.001
n.a.
F3, 32 = 3.59, P < 0.05
F3, 32 = 3.61, P < 0.05
n.a.
Dry matter content
F3, 26 = 12.56, P < 0.001
F3, 26 = 10.60, P < 0.001
n.a.
F3, 21 = 5.36, P < 0.01
F3, 21 = 2.08, P > 0.05
n.a.
n.a.
F3, 64 = 1.84, P > 0.05
F7, 64 = 3.96, P < 0.01
*
n.a.
*
F3, 64 = 0.39, P > 0.05
F7, 64 = 2.88, P < 0.05
Protein carbonyl content
F3, 64 = 231.71, P < 0.001
F3, 64 = 150.77, P < 0.001
F1, 64 = 85.57, P < 0.001
F3, 62 = 214.67, P < 0.001
F3, 62 = 129.51, P < 0.001
F1, 62 = 97.70, P < 0.001
Lipid peroxide content
F3, 64 = 160.15, P < 0.001
F3, 64 = 80.09, P < 0.001
F1, 64 = 58.17, P < 0.001
F3, 62 = 152.32, P < 0.001
F3, 62 = 78.11, P < 0.001
F1, 62 = 69.61, P < 0.001
Total ascorbate activity
F3, 64 = 189.02, P < 0.001
F3, 64 = 124.40, P < 0.001
F1, 64 = 55.07, P < 0.001
F3, 62 = 175.86, P < 0.001
F3, 62 = 91.45, P < 0.001
F1, 62 = 25.30, P < 0.001
Reduced ascorbate activity
F3, 63 = 87.10, P < 0.001
F3, 63 = 39.43, P < 0.001
F1, 63 = 5.35, P < 0.05
F3, 62 = 129.27, P < 0.001
F3, 62 = 50.96, P < 0.001
F1, 62 = 1.21, P > 0.05
Total glutathione activity
F3, 64 = 42.73, P < 0.001
F3, 64 = 35.13, P < 0.001
F1, 64 = 224.11, P < 0.001
F3, 62 = 38.42, P < 0.001
F3, 62 = 28.61, P < 0.001
F1, 62 = 261.31, P < 0.001
Reduced glutathione activity
F3, 64 = 320.06, P < 0.001
F3, 64 = 116.46, P < 0.001
F1, 64 = 2.97, P > 0.05
F3, 62 = 855.45, P < 0.001
F3, 62 = 282.62, P < 0.001
F1, 62 = 1.21, P > 0.05
Superoxide dismutase activity
F3, 64 = 108.32, P < 0.001
F3, 64 = 66.67, P < 0.001
F1, 64 = 9.11, P < 0.01
F3, 62 = 85.12, P < 0.001
F3, 62 = 48.76, P < 0.001
F1, 62 = 12.50, P < 0.01
Ascorbate peroxidase activity
F3, 64 = 39.25, P < 0.001
F3, 64 = 21.00, P < 0.001
F1, 64 = 19.18, P < 0.001
F3, 62 = 29.45, P < 0.001
F3, 62 = 22.81, P < 0.001
F1, 62 = 62.62, P < 0.001
Glutathione peroxidase activity
F3, 64 = 224.40, P < 0.001
F3, 64 = 85.59, P < 0.001
F1, 64 = 27.24, P < 0.001
F3, 62 = 191.28, P < 0.001
F3, 62 = 75.69, P < 0.001
F1, 62 = 2.21, P > 0.05
Catalase activity
F3, 64 = 209.43, P < 0.001
F3, 64 = 120.04, P < 0.001
F1, 64 = 11.65, P < 0.01
F3, 62 = 208.62, P < 0.001
F3, 62 = 104.22, P < 0.001
F1, 62 = 0.14, P > 0.05
F3, 64 = 2.57, P > 0.05
F3, 64 = 0.44, P > 0.05
F1, 64 = 225.941, P < 0.001
F3, 62 = 0.56, P > 0.05
F3, 62 = 1.35, P > 0.05
F1, 62 = 288.52, P < 0.001
F3, 64 = 94.80, P < 0.001
F3, 64 = 53.44, P < 0.001
F1, 64 = 26.81, P < 0.001
F3, 62 = 94.04, P < 0.001
F3, 62 = 56.81, P < 0.001
F1, 62 = 16.03, P < 0.001
Photosynthetic efficiency (Fv/Fm)
Markers of oxidative stress
Non-enzymatic antioxidants
Enzymatic antioxidants
Glutathione S-transferase
Glutathione reductase
a
Treatment: Uninfected / WClMV / Uninfected + Rf / WClMV + Rf
b
Time: day 0 - 2 - 5 - 7
c
Organ: Shoot vs Root
*
Fv/Fm values measured throughout the experiment
n.a. = not applicable
108
4.4 Discussion
In pastoral systems, root-flooding often occurs due to heavy rainfall and may significantly
reduce pasture growth in winter and early spring (McFarlane et al., 2007). As an important
component of pastures, white clover is prone to such abiotic stress regularly or at least
seasonally. Although T. repens has been reported to be relatively tolerant to excess water
(Pugh et al., 1995), the responses towards root-flooding when plants are infected with
viruses have not yet been, to our knowledge, studied. Accordingly, this experiment aimed
to determine how T. repens responded to a root-flooding treatment while infected with
WClMV, by assessing effects on growth parameters, oxidative damage and antioxidant
metabolism. If the plant dramatically suffers from both stress factors, the repercussions on
forage yield and pasture quality may be considerable.
Under soil flooding conditions, as low oxygen concentrations develop, anoxic tissues shift
their metabolism to rely on glycolytic and fermentative pathways, cellular energy being
drastically decreased (Bailey-Serres and Voesenek, 2008; Ferreira de Sousa and Sodek,
2002). Consequently, plant performance may be significantly affected (Huber et al., 2009),
and several studies reported flooding-induced inhibition of vegetative growth in different
plants (Przywara and Stepniewski, 1999; Yu and Rengel, 1999; Zhou and Lin, 1995). In the
current study, the two cultivars showed differences in growth under flooding stress. In
Huia, a decrease in fresh weight was observed when plants were waterlogged, with a more
severe effect when infected with WClMV. However, in Kopu II, the effect of waterlogging
was detrimental only in infected plants, virus-free plants displaying biomass accumulation
over the time experiment. Most importantly, in both cultivars, 8 days of root-flooding
treatment induced a significant reduction in dry matter content only in WClMV-infected
plants (refer to Fig. 39). This is a valuable observation for farmers as it indicates that, in the
field, excess water in soil and virus infection with WClMV can cause important yield loss
in T. repens in terms of food availability for livestock. Similar to the results shown in
Experiment 1 (refer to chapter 3, section 3.3.2.1), root-flooding alone, or in conjunction
with virus infection, did not impact the integrity of photosystem II apparatus as Fv/Fm was
not affected by both treatments. At first glance, this suggests that white clover may be
capable of maintaining its overall fitness to a certain level of tolerance. In this regard, it
109
should be noted over the time of this experiment, although plants displayed severe
symptoms, none had died off.
A sudden shift from aerobic to anaerobic conditions may exert great levels of stress and
result in the formation of ROS along with increased antioxidative defence mechanisms
(Irfan et al., 2010). In both cultivars, uninfected and WClMV-treated controls (i.e. nonwaterlogged) did not reveal any differences in oxidative stress parameters and antioxidant
metabolism measured in both shoots and roots. In Experiment 1, however, levels of
oxidative stress and the activity of antioxidants in infected plants were increased, the
consequence of which was attributed to both viral infection and harvesting. Such contrasts
may also have been dependent on the duration of the experiments, i.e. several weeks for
Experiment 1 vs a few days for Experiment 2. In the current experiment, only differences
between shoots and roots of non-waterlogged plants were observed. Indeed, the latter had
higher levels of protein carbonyl, lipid peroxide, total ascorbate, SOD, APX, GPX, CAT
and GR in shoots, and greater levels of total glutathione and GST in roots. Reduced
ascorbate/glutathione did not differ between organs. In contrast, root-flooding treatment
induced oxidative stress and changes in antioxidant metabolism in virus-free and WClMVinfected T. repens. Hypoxic conditions caused by excess water in soils are known to
generate oxidative stress along with ROS production in plant tissues, the consequences of
which strongly depend on species, plant tissues and the ability to trigger antioxidative
mechanisms (Sairam et al., 2008). The two cultivars used in this study showed similar
trends in the levels of oxidative damage and regulation of non-enzymatic and enzymatic
antioxidants that occurred in shoots and roots in response to flooding. Oxidative damage
was induced by flooding treatment as evidenced by increased levels of protein carbonyls
and lipid peroxides in both shoots and roots. Interestingly, it was greatly intensified in rootflooded plants that were also infected with the virus (Figs 41, 42, 43 & 44), indicating that
the interactive effect of both stresses can dramatically affect the clovers. As expected,
oxidative stress was more pronounced in roots than in shoots, most likely as a consequence
of direct O2-limitation effect taking place in the former.
110
Under root-flooding conditions, the modulation of antioxidant metabolism in plants with
up- and/or down-regulation of antioxidative components is likely to occur. A number of
studies have reported flooded plants with enhanced or diminished activities of antioxidants,
giving insight into the degree of tolerance that plants may display against hypoxic/anoxicinduced ROS formation (Kalashnikov et al., 1994; Lin et al., 2004; Yan et al., 1996;
Yordanova et al., 2004). In both cultivars, root-flooding induced an increase in total
ascorbate and glutathione contents, with higher glutathione levels found in roots. However,
as compared to uninfected-flooded plants, WClMV-treated clovers prone to waterlogging
showed less ascorbate and glutathione contents. In the attempt to maintain growth under the
harmful conditions caused by both stresses, the plant may allocate less energy towards the
synthesis of ascorbate and GSH, thereby impairing its ability to detoxify ROS.
Furthermore, the levels of both reduced ascorbate and reduced glutathione were decreased
in all parts of waterlogged plants (Figs 47, 48, 51 & 52). Accordingly, oxidation of
ascorbate and glutathione occurred in order to alleviate the effect of stress-induced ROS,
however, the regeneration of their reduced forms to maintain optimal intracellular redox
status was affected. Virus-infected plants prone to root-flooding were significantly more
affected in their ability to cope with oxidative damage, as reflected by decreased pools of
ascorbate/glutathione along with their reducing capacity.
In addition, the enzymatic activities of SOD, APX, GPX, CAT and GR were all upregulated in root-flooded plants, with greater activities found in plants that were not
infected with the virus (Figs 53 to 62). Interestingly, waterlogging did not induce or repress
GST activity, although roots displayed higher basal levels than shoots. These results
suggest that white clover can respond to flooding-caused oxidative stress by effectively
enhancing antioxidative metabolism. In this respect, T. repens seems to be able to tolerate
warterlogging to some extent. However, the effect that WClMV infection had on floodedplants should be emphasized. Indeed, increases in enzymatic antioxidant activities in
infected plants were significantly lower than in virus-free plants. Hence, it may be
hypothesized that the intensity of stress caused by both biotic and abiotic factors impacts
the plant in such a way that the majority of resources are used to sustain growth rather than
alleviate oxidative stress. Other studies have reported similar observations. Kumutha et al.
(2009) showed that, in a tolerant genotype of pigeon pea plant subjected to waterlogging
111
for 6 days, SOD, APX, CAT and GR activities were enhanced. In three different citrus
genotypes, oxidative stress in roots and leaves was induced by waterlogging, along with the
induction of ROS-scavenging antioxidants, the latter being more effective in plants
showing better tolerance (Arbona et al., 2008). In contrast, flooding-sensitive corn plants
grown under flooding conditions for 7 days displayed injury development as production of
active oxygen species was strongly favoured and antioxidant activities declined (Yan et al.,
1996).
Virus-free
Virus-free
WClMV
WClMV
LP + PC
LP + PC
↑ LP + PC
ASC
GSH
↑ Asc + GSH
SOD
SOD + APX
APX
GPX + CAT
CAT
GR
GR
ASC
↑ LP + PC
GSH
SOD↑ Asc + GSH
SOD + APX
APXGPX + CAT
CATGR
GR
↓ Red. Asc + Red. GSH
↓ Red. Asc + Red. GSH
Red. ASC
Red. GSH
Red. ASC
Red. GSH
Figure 63 Schematic representation of the levels of oxidative damage and antioxidants that were significantly
affected by root-flooding in virus-free and WClMV-infected T. repens (in both organs). Arrows show
increases or decreases, the size of which indicates major differences found between virus treatments. APX =
ascorbate peroxidase, ASC = ascorbate, CAT = catalase, GPX = glutathione peroxidase, GR = glutathione
reductase, GSH = glutathione, LP = lipid peroxidation, PC = protein carbonylation, Red. ASC = reduced
ascorbate,
Red.
GSH
=
reduced
glutathione,
SOD
=
superoxide
dismutase,
(picture
from
http://mycorrhizas.info/root.html).
112
Collectively, the current study indicates that root-flooding is an important abiotic stress that
may significantly affect the performance of T. repens. The treatment had a negative effect
on fresh weight accumulation in both cultivars, except for uninfected Kopu II plants.
Interestingly, the dry matter content of flooded plants decreased only in infected plants
(~60%). In this instance, root-flooding per se may not have any impact of DMC, but when
interacting with WClMV infection, the resulting effect may be detrimental for the plant. It
should be pointed out, however, that the experiment was conducted for 8 days only and that
over a longer period of flooding, uninfected plants might have displayed reduced DMC as
well. The ability of the plants to grow roots in the water and form aerenchymatous tissues
(Figs 35 & 36) would reflect a certain degree of tolerance towards flooding. These are
important traits of adaptation that should be selected for improving white clover tolerance
in the field. However, these newly developed roots lacked N2-fixing nodules. The absence
of such symbiotic association on newly developing roots would greatly impair the ability of
T. repens to fix atmospheric nitrogen, therefore decreasing its nutritive value and the
quality of the pasture. As expected, root-flooding induced oxidative stress in roots and
shoots, as confirmed by the increased levels of lipid peroxidation and protein carbonylation.
In order to counteract the adverse effects of ROS, white clover was able to trigger the
activities of antioxidants in all plant parts, although more intensively in roots in which
oxidative stress was stronger. Total ascorbate and glutathione levels were both enhanced,
although their reduced forms were diminished, indicating that high degree of oxidation
occurred in the cell. Except for GST, the activities of all enzymes, i.e. SOD, APX, GPX,
CAT and GR, were increased. However, when “double-stressed”, i.e. infected with
WClMV and root-flooded, the plants were significantly more sensitive and displayed
lessened non-enzymatic and enzymatic antioxidant activities in both shoots and roots. It is
not clear though, whether the down-regulation of ROS-scavenging mechanisms under rootflooding is a direct consequence of the virus, e.g. via repression of specific signal
transduction pathways, or whether it is due to the fact that the plant allocates more
resources towards growth. As cellular energy depletes under hypoxic conditions (Sairam et
al., 2008; Teakle et al., 2006), reduced resources must be available for the plant to be able
to cope with both stresses and maintain crucial metabolic pathways so that it does not
perish. Moreover, viruses are completely dependent on their host cell and must hijack the
cell’s machinery in order to replicate (Hull, 2009). It is therefore plausible that the energetic
113
cost associated with WClMV replication may restrict the amount of cellular energy
available for the host. Further research is needed to understand how the defence
mechanisms against oxidative damage, and the trade-offs between resources assigned to
growth/development and antioxidative metabolism, are established and regulated under
interaction between biotic (WClMV infection) and abiotic (root-flooding) stress in
Trifolium repens.
114
5. General discussion
5.1 Overview of the study
WClMV infection and excess water in soils (particularly in winter) are two important stress
factors likely to occur at high incidence in white clover pastures in New Zealand (Corkill et
al., 1981; Guy and Forster, 1996). Glasshouse trials conducted with two cultivars of
Trifolium repens provided relevant information on the effect of both biotic and abiotic
stresses on the legume plant’s performance.
White clover’s growing capacity was negatively affected by both stresses. The total aboveground biomass decreased under infection with WClMV, root-flooding treatment, and
under both conditions. Dry matter content was also significantly impacted. WClMV
infection induced a gradual reduction in DMC throughout the harvests in both cultivars,
although statistical differences were only found in Kopu II. However, given that Kopu II
was on average smaller in size than Huia at the beginning of the experiment, the trends in
DMC loss would probably be similar in Huia if further harvests had been conducted. In the
field, as grazing is likely to act as a continuous constraint for T. repens, the data presented
here suggests that the DMC of infected plants may dramatically decrease. This would result
in important losses in yield and feeding value of the pasture as a whole, hence affecting
feed intake of livestock.
WClMV infection and root-flooding induced oxidative stress in T. repens as evidenced by
increased levels of protein carbonyls and lipid peroxides, two important markers of
oxidative damage (Marrs, 1996; Moller et al., 2007). The appropriate induction of
antioxidative mechanisms is a key factor for removal of ROS and stress tolerance.
Ascorbate and glutathione are non-enzymatic low-molecular weight antioxidants
importantly involved in cell homeostasis maintenance by acting as redox buffer in the cell
(Apel and Hirt, 2004). Their antioxidative properties reside in their ability to be readily
oxidized by ROS, hence alleviating the effect of ROS oxidation on biological molecules
(Conklin, 2001; Noctor et al., 2012; Smirnoff, 1996). Accordingly, sufficient levels of
reduced ascorbate and glutathione must be sustained for efficient ROS-scavenging activity.
As presented in Experiment 2 though, the proportions of reduced forms were decreased by
115
the root-flooding treatment, suggesting that oxidation occurred at high levels without
optimal regeneration of reduced ascorbate and GSH. SOD is considered the first line of
defence against ROS via the detoxification of O2•- to H2O2, and is often up-regulated under
stress conditions (Apel and Hirt, 2004; Gill and Tuteja, 2010). APX and GPX are both
involved in the reduction of H2O2 using ascorbate and GSH, respectively, as electron
donors (Apel and Hirt, 2004; Gill and Tuteja, 2010; Shigeoka et al., 2002). Ascorbate and
GSH must then be regenerated to adequate levels for APX and GPX activities to be
monitored. CAT is found in all aerobic organisms and is responsible for the conversion of
H2O2 to water, without any requirement for reductants (Apel and Hirt, 2004; Mhamdi et al.,
2010; Scandalios et al., 1997). The role of GR in the ascorbate-glutathione cycle is to
catalyze the reduction of oxidized glutathione (GSSG) to GSH, using NADPH as reducing
equivalent (Ding et al., 2009; Gill and Tuteja, 2010). GSTs are enzymes that conjugate
GSH to electrophilic xenobiotic molecules that are potentially toxic for the cell (Dixon et
al., 2010; Marrs, 1996). In this study, in response to both stresses, levels of ascorbate and
glutathione, as well as the antioxidative enzymes SOD, APX, GPX, CAT and GR, were
increased. However, the levels of GST did not increase in response to WClMV infection or
flooding stress, indicating that the role of GSTs in responses to such stresses may not be
predominant. Additionally, according to pasture feed quality analyses, WClMV had no
major effect on the nutritional value of T. repens. This indicates that the plant can still
preserve its feed value per gram DM under infection, an important factor to take into
account for farming in terms of pastures value. When comparing both cultivars, Kopu II
appears to be more sensitive to WClMV infection as oxidative stress occurred at an earlier
stage of the experiment, which was consistent with the reduction in total biomass and the
onset of antioxidative mechanisms. However, the interesting feature of Kopu II over Huia
from a nutritional aspect is its higher nitrogen, mineral and crude protein content. On the
one hand, Huia seems to be a good candidate cultivar for pastoral management as it may be
more tolerant towards WClMV infection, but on the other hand, Kopu II may be judged to
be better in terms of nutritive value. As shown in Experiment 1, the harvesting per se might
impose a certain level of stress on the plants as it was strongly correlated to decreased plant
biomass, increased oxidative damage and enhancement of antioxidative activities. In the
root-flooding experiment, for which no harvesting effect could be assessed as all plant parts
(shoots and roots) were collected at each cutting, non-waterlogged controls did not differ in
116
their levels of oxidative stress and antioxidative activities. Hence, one may hypothesize that
the adverse effects of the virus on T. repens might have been favoured/amplified by
harvest-induced stress. If this is true, it is most likely to occur in pastoral systems via
livestock grazing or cropping equipment.
Flooding is a serious issue in terms of crop productivity in many regions (Jackson and
Colmer, 2005). To our knowledge, no studies have investigated the effect of the interaction
Virus x Root-flooding in plants. Arachevaleta et al. (1989) looked at the morphological and
physiological responses of the tall fescue (Festuca arundinacea) in mutualistic association
with the fungal endophyte (Acremonium coenophialum) under environmental stress,
including flooding. The endophyte protected tall fescue against drought stress but had no
effect on its host’s response to flooding. The current study emphasizes the negative
influence that flooding may have in plants suffering from virus infection. White clover has
been reported to be tolerant to root-flooding to some extent (Pugh et al., 1995), although
high variability in flooding tolerance are found between genotypes (Jackson et al., 2009).
Uninfected plants subjected to waterlogging experienced high degree of oxidative stress
over the time of the experiment, but were also capable of triggering antioxidant defence
mechanisms and maintaining growth. Nevertheless, the interactive effect of root-flooding
and WClMV infection was detrimental for the clovers, as indicated by reduced growth rate,
higher levels of oxidative damage and lower activity of antioxidant metabolism as
compared to uninfected-waterlogged plants. Plants may also suffer from post-hypoxia
oxidative stress, i.e. enhanced ROS production when oxygen is re-applied. Indeed, the
return in normoxic conditions (i.e. normal O2 concentrations) and sudden reactivation of
electron transport chain greatly favour the production of ROS (Irfan et al., 2010). Thus,
maintaining high levels of antioxidants during and following hypoxic conditions is a crucial
factor influencing plant tolerance to waterlogging (Kumutha et al., 2009). Flooding
tolerance may also be facilitated by the development of aerenchymas in root tissues, thus
improving gas exchange between aerated and anaerobic parts of the plant (Bailey-Serres
and Voesenek, 2008; Sairam et al., 2008). In this experiment, white clover displayed
aerotrophic adaptation, as evidenced by the formation of lysigenous aerenchymas in the
roots of flooded plants.
117
5.2 Future research
The current study provides entry points to novel promising areas of research that could
improve our perception of the mechanisms involved in plant-virus interactions. Little is
known of the effect of an additional abiotic stress on the interplay between hosts and
pathogens. In the present case, no literature concerning the impact of flooding on virusinfected plants has been documented, which would make this dissertation the first report
giving insight on such complex interactions.
The presence of WClMV in infected plants was confirmed by ELISA. However, the viral
titre of infected tissues was not determined. As future perspective, it would be relevant to
measure the viral titre of plant samples, using quantitative ELISA or qRT-PCR, to
demonstrate if other stresses (e.g. harvesting & root-flooding) have a significant impact on
virus replication in both roots and shoots. As white clover has been reported to be
susceptible to many plant viruses (see section 1.3.1), studying the effect of multiple
infections may represent another area of research. Another suggestion would be the design
of a realistic but practical root-environment that could improve the study of root systems
and facilitate their harvesting. Additional research needs to be undertaken to dissect the
responses behind tolerance or sensitivity of white clover towards WClMV and rootflooding. It would be pertinent to address the impact of post-hypoxia re-aeration on ROS
formation, as it may cause further oxidative damage in plant tissues; similarly, the effect of
elevated CO2 on WClMV infection should be addressed to determine if the disease could be
alleviated or worsened, with respect to predicted increases in atmospheric CO2 levels in the
upcoming century. Furthermore, the identification of potential PR genes/proteins in white
clover under WClMV infection by the use of genomic and proteomic techniques would
shed light on the resistance mechanisms involved during interaction. Finally, the use of
molecular tools to identify, for instance; quantitative trait loci (QTLs) associated with
resistance traits, anaerobic proteins (ANPs) expressed under hypoxic/anoxic conditions
(such as the alcohol dehydrogenase), the metabolic pathways involved in resistance and
ROS formation under flooding, may contribute to our understanding of the processes
involved in such interactions and help to produce/engineer new T. repens cultivars with
greater tolerance to environmental stress conditions.
118
5.3 Conclusions
This thesis has aimed to shed light on T. repens performance under environmental stress
conditions. The data indicates that white clover may cope relatively well with WClMV
infection, but when subjected to other stresses, such as harvesting or root-flooding, its
fitness may be reduced. Plants may respond differently to multiple stresses from than to
individual stresses, in a positive or negative way depending on the interactions (Atkinson
and Urwin, 2012). For instance, elevated CO2 alleviated the effect of the root parasite
Orobanche minor on T. repens growth (Dale and Press, 1998) and the effect of BYDV in
oats (Malmstrom and Field, 1997), while it enhanced bacterial disease in chilli pepper
plants (Shin and Yun, 2010).
This study may contribute to improve pastoral management by pointing out the importance
of stress factors on pasture plant performance and herbage yield. WClMV infection and
root-flooding are likely to significantly impact biomass accumulation in the field, making
white clover less able to compete with other pasture plants. Accordingly, a decrease in the
nutritional value of pastures is an inevitable outcome, as nitrogen assimilation by clovers
would be greatly affected. Hence, in the attempt of compensating this loss of pastoral
quality, it may represent a considerable cost for farmers in terms of nitrogen input. The
importance of selecting morphological and physiological traits for improving white clover
tolerance against biotic and abiotic stress must then be emphasized. In the context of this
study, the development of new cultivars via breeding programs for introducing virus
resistance in clovers grown in waterlogged regions should be considered. Although Kopu II
has been reported to outperform Huia in New Zealand grazed pastures (Woodfield et al.,
2001), from our results, Huia may be considered a better candidate for pastoral farming
over Kopu II as it displayed better tolerance against WClMV infection. Both cultivars
showed high phenotypic variability during the experiments. The experimental design could
have been improved if these phenotypic differences were taken into account in the
sampling method. Indeed, it would have been wise to sample the plants according to size
class, e.g. small – medium – large. In this way, the effect of phenotype would have been
lessened, and the assessment of the effect of WClMV infection would have shown less
variation and enabled better identification of the degree of interaction.
119
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