Journal of Experimental Botany, Vol. 63, No. 12,
2, pp.
2012
pp.695–709,
4321–4331,
2012
doi:10.1093/jxb/err313
doi:10.1093/jxb/ers143 Advance
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publication 44 November,
June, 20122011
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RESEARCH
PAPER
REVIEW PAPER
In
Posidonia
oceanica
cadmium
induces
changes
in DNA
Foliar
pathogenesis
and
plant water
relations:
a review
methylation and chromatin patterning
Michael K. Grimmer,1,* M. John Foulkes2 and Neil D. Paveley3
1
ADAS Greco,
UK Ltd, Battlegate
Rd, Boxworth, Leonardo
Cambridge, Bruno
Cambridgeshire
CB23Beatrice
4NN, UK Bitonti*
Maria
Adriana Chiappetta,
and Maria
2
University of Nottingham, Sutton Bonington Campus, Loughborough, Leicestershire LE12 5RD, UK
Department
of Ecology,
University Duggleby,
of Calabria,Malton,
Laboratory
Plant Cyto-physiology,
3
ADAS UK Ltd,
High Mowthorpe,
NorthofYorkshire
YO17 8BP, UK Ponte Pietro Bucci, I-87036 Arcavacata di Rende,
Cosenza, Italy
* To whom correspondence should be addressed. E-mail: [email protected]
* To whom correspondence should be addressed. E-mail: [email protected]
Received 2 April 2012; Revised 9 May 2012; accepted 6 February 2012
Received 29 May 2011; Revised 8 July 2011; Accepted 18 August 2011
Abstract
Abstract
As the world population grows, there is a pressing need to improve productivity from water use in irrigated and rainIn mammals, cadmium is widely considered as a non-genotoxic carcinogen acting through a methylation-dependent
fed agriculture. Foliar diseases have been reported to decrease crop water-use efficiency (WUE) substantially, yet
epigenetic mechanism. Here, the effects of Cd treatment on the DNA methylation patten are examined together with
the effects of plant pathogens are seldom considered when methods to improve WUE are debated. We review the
its effect on chromatin reconfiguration in Posidonia oceanica. DNA methylation level and pattern were analysed in
effects of foliar pathogens on plant water relations and the consequences for WUE. The effects reported vary between
actively growing organs, under short- (6 h) and long- (2 d or 4 d) term and low (10 mM) and high (50 mM) doses of Cd,
host and pathogen species and between host genotypes. Some general patterns emerge however. Higher fungi and
through a Methylation-Sensitive Amplification Polymorphism technique and an immunocytological approach,
oomycetes cause physical disruption to the cuticle and stomata, and also cause impairment of stomatal closing
respectively. The expression of one member of the CHROMOMETHYLASE (CMT) family, a DNA methyltransferase,
in the dark. Higher fungi and viruses are associated with impairment of stomatal opening in the light. A number of
was also assessed by qRT-PCR. Nuclear chromatin ultrastructure was investigated by transmission electron
toxins produced by bacteria and higher fungi have been identified that impair stomatal function. Deleterious effects
microscopy. Cd treatment induced a DNA hypermethylation, as well as an up-regulation of CMT, indicating that de
are not limited to compatible plant–pathogen interactions. Resistant and non-host interactions have been shown to
novo methylation did indeed occur. Moreover, a high dose of Cd led to a progressive heterochromatinization of
result in stomatal impairment in light and dark conditions. Mitigation of these effects through selection of favourable
interphase nuclei and apoptotic figures were also observed after long-term treatment. The data demonstrate that Cd
resistance responses could be an important breeding target in the future. The challenges for researchers are to
perturbs the DNA methylation status through the involvement of a specific methyltransferase. Such changes are
understand how the effects reported from work under controlled conditions translate to crops in the field, and to
linked to nuclear chromatin reconfiguration likely to establish a new balance of expressed/repressed chromatin.
elucidate underlying mechanisms.
Overall, the data show an epigenetic basis to the mechanism underlying Cd toxicity in plants.
Key words: Photosynthesis, plant pathogens, resistance, stomatal conductance, transpiration, water-use efficiency.
Key words: 5-Methylcytosine-antibody, cadmium-stress condition, chromatin reconfiguration, CHROMOMETHYLASE,
DNA-methylation, Methylation- Sensitive Amplification Polymorphism (MSAP), Posidonia oceanica (L.) Delile.
Introduction
Introduction
Plants lose water from leaves due to a large gradient in water tightly enough to increase the stomatal resistance to water loss
potential
between foliar mesophyll
cells and the
levels comparable
with thatfor
of the
cuticle.
The stomatal
aperIn
the Mediterranean
coastal ecosystem,
theatmosphere
endemic to Although
not essential
plant
growth,
in terrestrial
(Ayres, 1986).
The gradient
soil plays
and leaves
is considtures areCd
adjusted
in intricate
diurnal
cyclesand
to maintain
a balance
seagrass
Posidonia
oceanicabetween
(L.) Delile
a relevant
role plants,
is readily
absorbed
by roots
translocated
into
erably
smaller primary
and so theproduction,
gradient at the
leaf–air
interface (plus
in plantorgans
water while,
relations.
In the absence
stress,taken
stomata
by
ensuring
water
oxygenation
and aerial
in acquatic
plants,ofit water
is directly
up
solar radiation/sensible
heat for
energy) besides
provides counteracting
the main driv- by
in most
plants
open in
light andinduces
close incomplex
the dark.changes
Under
provides
niches for some
animals,
leaves.
In plants,
Cdthe
absorption
ing forceerosion
for water
movement.
At this interface,
a hydrophobic
water
stomatal
closure can
as a rapid levels
and effective
coastal
through
its widespread
meadows
(Ott, 1980; at
thestress,
genetic,
biochemical
andserve
physiological
which
cuticle covers
outer
surface ofetepidermal
cellsThere
and isishighly
drought resistance
via dehydration
avoidance.
Piazzi
et al., the
1999;
Alcoverro
al., 2001).
also ultimately
accountresponse
for its toxicity
(Valle and
Ulmer, Lower
1972;
resistant to water
loss. The
is punctuated
by stomatal
stomataldiconductance
improves 1999;
instantaneous
water-use
efficonsiderable
evidence
that cuticle
P. oceanica
plants are
able to Sanitz
Toppi and Gabrielli,
Benavides
et al., 2005;
pores through
which CO2 for
photosynthesis
diffuses(Sanchiz
into the Weber
ciency (WUE)
part by
a higherThe
concentration
graabsorb
and accumulate
metals
from sediments
et al., in2006;
Liufavouring
et al., 2008).
most obvious
leafal.,
and
water
vapour diffuses out
intoMaserti
the atmosphere.
Thethus
sto- symptom
dient of CO
the is
airaoutside
and in
inside
leaf (Condon
et
1990;
Pergent-Martini,
1998;
et al., 2005)
of2 between
Cd toxicity
reduction
plantthegrowth
due to
matal pores are
eachbioavailability
flanked by a pairinofthe
guard
cells which
regu- an
et al.,
2004); overall,
as stomatal conductance
decreases,
there is
influencing
metal
marine
ecosystem.
inhibition
of photosynthesis,
respiration,
and nitrogen
late the
aperture
therefore
the rate considered
at which water
is metabolism,
a greater impact
diffusion
of water outinofwater
the stomata
than on
For
thispore
reason,
thisand
seagrass
is widely
to be
as on
well
as a reduction
and mineral
frombioindicator
the leaf. The species
guard cells
can close
the1988;
pore aperture
the rate (Ouzonidou
of photosynthesis.
prolonged stomatal
alost
metal
(Maserti
et al.,
Pergent uptake
et al.,However,
1997; Perfus-Barbeoch
et al.,closure
2000;
et al., 1995; Lafabrie et al., 2007). Cd is one of most Shukla et al., 2003; Sobkowiak and Deckert, 2003).
widespread
heavy metals in both terrestrial and marine
At the genetic level, in both animals and plants, Cd
Abbreviations: A, CO2 assimilation rate; E, transpiration rate; WUE, water-use efficiency (above-ground dry matter produced per unit of water transpired and
environments.
canproduced
induce
chromosomal
aberrations,
abnormalities
in
evaporated per unit land area); WUET, transpiration efficiency (above-ground dry matter
per unit
of water transpired
per unit land area);
WUEI, instantaneous
water-use efficiency ( A/E at the leaf level).
© 2011
The Author
[2012]. Published by Oxford University Press [on behalf of the Society for Experimental Biology]. All rights reserved.
ª
The Author(s).
For Permissions,
please article
e-mail:distributed
[email protected]
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is an Open Access
under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/bync/3.0), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
4322 | Grimmer et al.
is not sustainable, as CO2 uptake is also reduced and will ultimately limit photosynthetic assimilation and growth (Farquhar and
Sharkey, 1982; Schulze et al., 1987).
Since early work on crown rust (Puccinia coronata Corda) of
oats (Avena sativa L.) by Murphy (1935), a wide range of foliar
pathogens have been shown to disrupt the cuticular or stomatal
regulation of transpiration and therefore affect plant water relations (reviewed by Ayres, 1981). The strategies for direct foliar
infection and propagule exit employed by a range of pathogen
types are summarized in Table 1, and indicate various ways in
which cuticular or stomatal resistance to water loss can be compromised. First, when pathogens invade the leaf they can either
pass through the stomatal pores or breach the cuticle. Secondly,
pathogens may produce spore-bearing structures from the leaf
that can either pass through the stomatal pores or erupt through
the epidermis, tearing open the cuticle. Sporulation of rust pathogens in particular is associated with cuticular breach. Thirdly,
infections can occur within the substomatal cavity and affect
stomatal function. Finally, both pathogenesis and host resistance
responses can result in an alteration in guard cell regulation of stomatal aperture. We review these effects of foliar pathogenesis on
leaf water relations and highlight the implications for crop WUE.
Water-use efficiency
As the world population grows, there are increasing demands
on water supply for domestic, industrial, and agricultural uses.
Of the world’s available water resource, ~80% is currently consumed by irrigated agriculture (Condon et al., 2004). Rain-fed
agriculture, however, is the primary means of food production
in most countries, including the majority of dryland countries.
Most of the water which falls on dryland crops is returned to
the atmosphere by evapotranspiration and fails to reach rivers
and aquifers. Human intervention in natural water cycles through
cropping can therefore fundamentally affect ground water supplies and landscape hydrology. Climate change is imposing further perturbations, altering seasonal precipitation patterns, and
reducing total availability (Jenkins et al., 2009). In light of these
pressures, several strategies will be required to improve the productivity of water use in irrigated and rain-fed agriculture. Considerable efforts have already been made to improve rainfall-use
efficiency by agronomic practice (reviewed by Turner, 2004) and
to improve WUE by plant breeding (reviewed by Condon et al.,
2004; Cattivelli et al., 2008), but the effects of plant pathogens
have not been considered adequately in this context.
The term water-use efficiency (g DM l–1) can be defined as the
total amount of above-ground dry matter produced per unit of
water transpired and evaporated per unit of land area. The term
transpiration efficiency (WUET; g DM l–1) can be defined as the
total amount of dry matter produced per unit of water transpired
per unit of land area. Instantaneous water-use efficiency (WUEI)
is the ratio of CO2 assimilation rate to transpiration rate at the
leaf level. WUEI does not always correspond to WUET over a
season as there are many processes integrated over the growing
season. WUEI forms an important measure of the sustainability
of crop production and depends both on cuticular integrity and
normal guard cell regulation of stomatal aperture in response to
solar radiation, the partial pressure of CO2, and water availability
(Condon et al., 2004).
An early study by Murphy (1935) showed that crown rust infection reduced the WUET of glasshouse-grown oats by 50%. In two
susceptible varieties infected from the seedling stage, the average of above-ground dry matter was 38% that of the uninfected
controls. However, the amount of water used to maintain 85%
soil moisture was 68% that of controls, so WUET was reduced
from 3.1 g DM l–1 to 1.7 g DM l–1 by the crown rust infection.
These figures refer to the total above-ground dry matter, whereas
the trait of agronomic importance is grain yield. In the Murphy
(1935) example, the average harvest index (HI; the ratio of grain
dry mass to above-ground dry mass) for infected plants and for
uninfected controls was 0.02 and 0.40, respectively. Therefore,
the WUET (grain) was reduced from 0.88 g l–1 to 0.04 g l–1 by
the crown rust infection, a decrease of ~95%. Although these
results were derived from an infection level which is unlikely to
occur in the field under normal cultural practices, they highlight
the potential for waste of water resources through foliar disease.
Even following a relatively late crown rust infection at anthesis,
WUET (grain) was reduced by 40% (Murphy, 1935).
Partial closure of the stomata during mild drought stress in
healthy plants increases the concentration gradient of CO2 from
the air into the leaf relatively more than it increases the concentration gradient of water vapour out of the leaf to the air, therefore
favouring higher WUEI. The gradients alone, however, do not
explain the effect on WUEI. Importantly, CO2 assimilation nor-
Table 1. Strategies for direct foliar infection and propagule exit of a range of plant pathogen types
Pathogen
Kingdom
Order
Typical foliar infection method
Typical propagule exit method
Powdery mildews
Fungi
Erysiphales
Rusts
Fungi causing
necrotic lesions
Oomycetes
Fungi
Fungi
Pucciniales
Various
Chromista
Various
Bacteria
Bacteria
Various
Viruses
–
Various
Penetrate directly through
epidermis
Penetrate via stomata
Penetrate directly through
epidermis or via stomata
Penetrate directly through
epidermis or via stomata
Enter leaf through stomata
or foliar wounds
Direct entry via feeding of
insects and mites
Spore-bearing structures
form on leaf surface
Epidermis ruptures to release spores.
Epidermis breached by
spore-bearing structures
Spore-bearing structures
emerge through stomata
Bacterial cells exude from
stomata or from lesions
Direct exit via feeding of
insects and mites
Foliar pathogenesis and plant water relations | 4323
mally increases non-linearly with stomatal conductance (since
internal CO2 becomes saturating for photosynthesis), whereas
transpiration increases linearly with stomatal conductance. Paul
and Ayres (1984) imposed water restrictions on common groundsel (Senecio vulgaris L.) plants in a controlled environment
and WUEI increased. This response was inhibited, however, in
plants infected by rust (Puccinia lagenophorae Cooke). Nus
and Hodges (1986) looked at the effect of stripe smut (Ustilago
striiformis West.) on WUEI in smooth meadowgrass (Poa pratensis L.) under controlled conditions. In leaves of plants exhibiting moderate to heavy stripe smut sporulation and subsequent
cuticle damage, WUEI was decreased by ~32% compared with
leaves of healthy plants. Further studies have identified reductions in WUEI caused by fungal diseases of grapevine (Vitis L.;
Lakso et al., 1982), pecan [Carya illinoinensis (Wang.) Koch;
Andersen et al., 1990], and common bean (Phaseolus vulgaris
L.; Jesus Junior et al., 2001) using leaf gas exchange equipment.
In contrast, Radwan et al. (2006) found no significant difference in WUEI between healthy leaves of pumpkin (Cucurbita
pepo L.) and leaves infected with Zucchini yellow mosaic virus
Lisa, despite significant reductions in stomatal conductance (gs)
and transpiration rate (E). Radwan et al. (2008) also reported no
change in WUEI for field bean (Vicia faba L.) leaves infected
with Bean yellow mosaic virus Dool. & Jones, with reductions
in AN balanced by similar reductions in E. However, these WUEI
measurements were taken under light conditions and therefore
did not test for the potential loss of water vapour in the dark, if
deregulated stomata remained partially open.
Shtienberg (1992) used gas-exchange equipment to investigate the effects of foliar disease on photosynthesis and transpiration in 10 different pathosystems in the field. Regardless of the
pathosystem, photosynthesis was affected in a similar manner
(i.e. a linear decrease with increments of the natural logarithm
of disease severity). In addition, the reduction was greater than
would be expected on the basis of the reduction in the proportion of healthy leaf area; that is, through changes in leaf area
modifying fractional interception of radiation according to the
equation of Monsi and Saeki (1953), assuming no change in
the light extinction coefficient (K) or radiation-use efficiency
(above-ground dry matter/radiation interception). The relative transpiration rate decreased with increasing disease severity, with the amount of decrease varying substantially between
pathosystems. These variations were due in some part to the type
of trophic relationship between pathogen and host. At low levels
of infection (severity <10%), rusts in maize (Zea mays L.) and
wheat (Triticum aestivum L.) increased transpiration compared
with disease-free controls. At higher rust severities transpiration
was reduced, but the reduction was smaller than expected based
on the proportion of leaf area affected. Fungi causing necrotic
lesions in cotton (Gossypium barbadense L.), wheat, and mango
(Mangifera indica L.) reduced transpiration, with rates close
to values expected. Powdery mildews of wheat, peach [Prunus
davidiana (Carr.) Franch.], and grapevine (V. vinifera) reduced
transpiration more than would be expected on the basis of the
proportional reduction in leaf area. It could be inferred from
Shtienburg’s findings that WUEI in the light would be expected
to be decreased by rusts, increased by powdery mildews, and
relatively unchanged by fungi causing necrotic lesions.
Techniques for measuring the stomatal
aperture size and function
Since stomata have a critical role in foliar pathogenesis and
plant water relations, it is important that accurate measurements of stomatal aperture characteristics are obtained in order
to investigate guard cell function. Various measurement methods have been described, including: (i) visual observation of
individual stomata by microscopy and measurement of actual
or relative aperture sizes (Hewitt and Ayres, 1975; Guimarães
and Stotz, 2004; Shafiei et al., 2007), which has been shown
to correlate with indirect measures of stomatal aperture (e.g.
Scharte et al., 2005; Allègre et al., 2007); (ii) the use of porometers or gas-exchange equipment to determine the rate at which
water is lost from a given area of leaf tissue, known as the
leaf conductance to water vapour loss (gl) (Hsiao et al., 1975;
Rebetzke et al., 2000); and (iii) thermal imaging to detect spatial and temporal changes in stomatal apertures in plant–pathogen interactions. The last is possible since there is a negative
correlation between evaporation rate, due to water loss through
the stomata, and leaf temperature (Inoue et al., 1990). This
technique has been used to detect diseased regions of the leaf
before visual symptoms were apparent (Lindenthal et al., 2005;
Oerke et al., 2006) and to identify infections in asymptomatic
leaves (Chaerle et al., 2006).
Effects of pathogenesis and host resistance
on stomatal function
A literature search was conducted to investigate evidence for
the effects of foliar pathogenesis and host resistance on stomatal
function. Pathogens which only infect roots or stems were omitted from the search as it was considered that they would affect
hydraulic conductance of water through the plant rather than
exert a local influence on stomatal guard cells.
Impairment of stomatal opening
For a wide range of pathosystems it was found that stomatal
aperture size in the light was reduced compared with healthy
controls (Table 2). The only exceptions to this were leaves
infected with oomycete pathogens, where stomatal aperture in
the light increased—discussed in a later section. The examples
in Table 2 include a wide range of dicotyledonous crop species
and the monocotyledonous crops maize and barley (Hordeum
vulgare L.). A diverse range of plant pathogens are represented,
including biotrophic, necrotrophic, and hemibiotrophic fungi
and viruses.
A number of mechanisms have been proposed by which
stomatal opening in the light may be impaired during pathogenesis. Lindsey and Gudauskas (1975) suggested that stomata
within chlorotic regions of maize leaves, following infection
with Maize dwarf mosaic virus Will. & Alex., were less functional than those in greener areas of infected leaves. It was
proposed that the reduction in chlorophyll content associated
with infection was responsible for decreased stomatal conductance, since chlorophyll pigments are a prerequisite for stomatal
4324 | Grimmer et al.
Table 2. Pathosystems in which stomatal aperture decreases in the light have been demonstrated
Host
Host scientific name
Pathogen
Trophic typea
Test environment
Methodb
Reference
Sugar beet
English oak
Beta vulgaris L.
Quercus robur L.
–
Biotroph
Field
Glasshouse
GE
GE
Maize
Zea mays L.
–
Controlled
M
Pea
Barley
Pisum sativum L.
Hordeum vulgare L.
Biotroph
Biotroph
Controlled
Controlled
P
P
Sugar beet
Beta vulgaris L.
Biotroph
Controlled
GE
Pecan
Hemibiotroph
Field
GE
Hall and Loomis (1972)
Hewitt and Ayres
(1975)
Lindsey and
Gudauskas (1975)
Ayres (1976)
Ayres and Zadoks
(1979)
Gordon and Duniway
(1982a)
Andersen et al. (1990)
Field bean
Carya illinoinensis
(Wang.) Koch
Vicia faba L.
Beet yellows virus Roland
Erysiphe alphitoides (Griffon & Maubl.)
Braun & Takam
Maize dwarf mosaic virus Will.
& Alex.
Erysiphe pisi DC.
Blumeria graminis (DC.)
Speer f.sp. hordei
Erysiphe betae
(Vaňha) Weltzien
Mycosphaerella dendroides
(Cooke) Demaree & Cole
Botrytis fabae Sardiña
Necrotroph
Glasshouse
GE
Sweet cherry
Prunus avium L.
Blumeriella jaapii (Rehm) Arx
Hemibiotroph
Glasshouse
GE
Sour cherry
Prunus cerasus L.
Blumeriella jaapii (Rehm) Arx
Hemibiotroph
Glasshouse
GE
Sugar beet
Common bean
Beta vulgaris L.
Phaseolus vulgaris L.
–
Hemibiotroph
Glasshouse/field
Field
GE
GE
Common bean
Phaseolus vulgaris L.
Biotroph
Phaseolus vulgaris L.
Glasshouse/
outdoor
Glasshouse/
outdoor
GE
Common bean
Beet yellows virus Roland
Phaeoisariopsis griseola
(Sacc.) Ferraris
Uromyces appendiculatus
Strauss
Colletotrichum lindemuthianum
(Sacc. & Magnus) Briosi
& Cavara
Common bean
Phaseolus vulgaris L.
Hemibiotroph
Controlled
GE
Meyer et al. (2001)
Hemibiotroph
Controlled
GE
Bassanezi et al. (2002)
Hemibiotroph
Controlled
GE
Bassanezi et al. (2002)
–
–
Field/outdoor
Controlled
GE
P/M
Sampol et al. (2003)
Bertamini et al. (2004)
Hemibiotroph
Field
GE
–
–
Controlled
Glasshouse
GE
GE
Roloff and Scherm
(2004)
Zhou et al. (2004)
D.-P. Guo et al. (2005)
–
Glasshouse
GE
Y.-P. Guo et al. (2005)
Biotroph
Field
GE
Moriondo et al. (2005)
–
Controlled
GE/M/TH
Chaerle et al. (2006)
Hemibiotroph
Field
GE
Biotroph
Controlled
P/M
Pinkard and
Mohammed (2006)
Prats et al. (2006)
–
Controlled
GE
Radwan et al. (2006)
Hemibiotroph
Outdoor
GE
Biotroph
Outdoor
GE
Ponmurugan et al.
(2007)
Ponmurugan et al.
(2007)
Common bean
Common bean
Grapevine
Grapevine
Blueberry
Potato
Stem mustard
Radish
Grapevine
Tobacco
Eucalyptus
Barley
Pumpkin
Tea
Tea
Colletotrichum lindemuthianum (Sacc.
& Magnus) Briosi
& Cavara
Phaseolus vulgaris L.
Phaeoisariopsis griseola
(Sacc.) Ferraris
Phaseolus vulgaris L.
Colletotrichum lindemuthianum (Sacc.
& Magnus)
Briosi & Cavara
Vitis vinifera L.
Three different virusesc
Vitis vinifera L.
Grapevine leafroll-associated virus-3
Cand. & Mart.
Vaccinium L. spp.
Septoria albopunctata
Cooke
Solanum tuberosum L.
Potato virus Y Smith
Brassica juncea (L.) Czern. Turnip mosaic virus
Schultz
Raphanus sativus L.
Turnip mosaic virus
Schultz
Vitis vinifera L.
Uncinula necator
(Schwein.) Burrill
Nicotiana tabacum L.
Pepper mild mottle virus
McKinney
Eucalyptus globulus Labill. Mycosphaerella
Johanson spp.
Hordeum vulgare L.
Blumeria graminis (DC.)
Speer f.sp. hordei
Cucurbita pepo L.
Zucchini yellow mosaic
virus Lisa
Camellia sinensis (L.)
Exobasidium vexans
Kuntze
Massee
Camellia sinensis (L.)
Pestalotiopsis theae
Kuntze
(Sawada) Steyaert
Hemibiotroph
GE
Malthus and Madiera
(1993)
Niederleitner and
Knoppik (1997)
Niederleitner and
Knoppik (1997)
Clover et al. (1999)
Jesus Junior et al.
(2001)
Lopes and Berger
(2001)
Lopes and Berger
(2001)
Foliar pathogenesis and plant water relations | 4325
Continued
Table 2. Pathosystems
in which stomatal aperture decreases in the light have been demonstrated
Host
Host scientific name
Pathogen
Trophic typea
Test environment
Methodb
Reference
Tea
Outdoor
GE
Biotroph
Outdoor
GE
Biotroph
Biotroph
Hemibiotroph
Controlled
Controlled
Field
P
P
GE
–
Controlled
GE
Ponmurugan et al.
(2007)
Ponmurugan et al.
(2007)
Prats et al. (2007)
Prats et al. (2007)
Premkumar et al.
(2008)
Radwan et al. (2008)
English oak
Quercus robur L.
Colletotrichum coccodes
(Wallr.) Hughes
Cephaleuros virescens
Kunze
Puccinia hordei Otth
Puccinia triticina Erikss.
Exobasidium vexans
Massee
Bean yellow mosaic virus
Dool. & Jones
Erysiphe alphitoides
(Griffon & Maubl.) Braun &
Takam
Hemibiotroph
Field bean
Camellia sinensis (L.)
Kuntze
Camellia sinensis (L.)
Kuntze
Hordeum vulgare L.
Triticum aestivum L.
Camellia sinensis (L.)
Kuntze
Vicia faba L.
Biotroph
Glasshouse
GE
Hajji et al. (2009)
Tea
Barley
Wheat
Tea
a
b
c
Viral pathogens not attributed with a trophic type.
Detection methods are gas exchange (GE), microscopy (M), porometry (P), and thermography (TH).
Grapevine leaf roll-associated virus Cand. & Mart., Grapevine fan leaf virus Rathay, and Grapevine fleck virus Boscia.
opening in the light (Virgin, 1957). Meyer et al. (2001) also
proposed that damage to the photosynthetic apparatus resulted
in stomatal closure, in studies of common bean leaves infected
by Colletotrichum lindemuthianum (Sacc. & Magnus) Briosi
& Cavara. Tentoxin, a toxin produced by Alternaria alternata (Fr.) Keissl., inhibits chloroplastic ATPase (Steele et al.,
1976) and induces an irreversible stomatal closure (Dahse et
al., 1990). As well as effects due to photosynthetic impairment,
stomatal aperture size is known to be reduced by a number of
compounds that accumulate during pathogenesis, such as salicylic acid (Chaerle et al., 1999), nitric oxide (NO) (Neill et al.,
2002; Del Rio et al., 2004; Mur et al., 2005), phenolic compounds (Plumbe et al., 1986), and the plant hormones abscisic
acid (ABA) and auxin (Whenham and Fraser, 1981; Grabov
and Blatt, 1998).
To determine whether these effects impact substantially on
crop growth ideally requires that the impairment is shown to
occur under natural conditions. Although many of the examples in Table 2 result from controlled environment work,
in a number of studies a crop was grown and tested in the
field. For all of the pathosystems analysed in the field there
were reported reductions in CO2 assimilation rate and/or photosynthetic rate associated with the reductions in stomatal
conductance. It is important, however, to determine whether
stomatal conductance is the cause of these assimilation and
photosynthesis reductions or whether it is a symptom of a
general impairment of photosynthesis. For the grapevine (V.
vinifera)–virus (Sampol et al., 2003) and eucalyptus (Eucalyptus globulus Labill.)–Mycosphaerella Johanson spp. (Pinkard and Mohammed, 2006) pathosystems, it was concluded
that pathogen-associated reductions in photosynthesis were
due to reduced activity of ribulose-1,5-bisphosphate carboxylase-oxygenase (RuBisCo), which catalyses the first major
step of carbon fixation, with reductions in mesophyll conductance to CO2 also implicated. For the sugar beet–Beet yellows
virus pathosystem (Clover et al., 1999), it was suggested that
the reduction in stomatal conductance probably resulted from
the decrease in photosynthetic rate rather than vice versa.
However, for the pecan–Mycosphaerella dendroides (Cooke)
Demaree and Cole (Andersen et al., 1990) and grapevine
(V. vinifera)–Grapevine leafroll-associated virus-3 Cand. &
Mart. (Bertamini et al., 2004) pathosystems, the reductions
in stomatal conductance were closely linked with decreases in
photosynthetic processes, suggesting that the photosynthetic
rate was tightly regulated by the stomatal control of CO2 conductance. It appears that the extent to which reductions in
stomatal conductance limit the photosynthetic rate of infected
leaves depends on the particular pathosystem in question. This
is also borne out by analysis of the literature on pathosystems
tested under controlled conditions. It is difficult, therefore, to
make general conclusions about whether pathogen-associated
reductions in stomatal conductance are the direct cause of
photosynthetic decline or an indirect effect of reduced photosynthetic rate.
Impairment of stomatal closure
The impairment of stomatal closure in the dark during foliar
pathogenesis has been reported for powdery mildew disease of
pea (Pisum sativum L.) (Ayres, 1976), barley (Ayres and Zadoks,
1979), and sugar beet (Gordon and Duniway, 1982b). However,
no direct observations of stomata were made in these studies,
and so the contribution of the fungal mycelium to the decrease
in leaf resistance to water loss could not determined. Prats
et al. (2006) found no significant difference in stomatal conductance in the dark between healthy spring barley leaves and
leaves infected with the biotroph Blumeria graminis (DC.)
Speer f.sp. hordei. This study looked at young first leaves,
whereas Ayres and Zadoks (1979) observed effects on mature
flag leaves. This difference may be relevant because stomatal
conductance in healthy leaves is related to leaf age and thus
4326 | Grimmer et al.
to ageing of the stomatal apparatus (Wardle and Short, 1983;
Willmer et al., 1988). Another distinction is that Prats et al.
(2006) observed barley leaves in the first 2 d after inoculation
before visual symptoms would have occurred, whereas Ayres
and Zadoks (1979) observed effects after visual symptoms had
appeared.
Ayres and Jones (1975) observed stomatal apertures in barley leaves infected by the hemibiotroph Rhynchosporium secalis (Oudem.) Davis and found that as the infection progressed
towards sporulation, an increasing percentage of stomata
failed to close in the dark compared with healthy leaves. It
was suggested that the stomatal opening might have been due
to cytokinins which are produced by R. secalis in culture and
accumulate in leaves infected by R. secalis. Digby and Cooper
(1972) showed that the cytokinin kinetin enhances stomatal
opening in barley.
Guimarães and Stotz (2004) identified that oxalate produced
by the necrotroph Sclerotinia sclerotiorum (Lib.) de Bary caused
the stomata of field beans to remain open in the dark following inoculation of the adaxial leaf surface. The fungus exploited
open stomatal pores to emerge from the uninoculated abaxial
leaf surface. Oxalate appeared to be an important pathogenicity
determinant, as susceptibility was reduced in an oxalate-deficient fungal mutant. ABA-induced stomatal closure of healthy
leaves was significantly reduced by treatment with oxalate, indicating that stomata of diseased leaves would be impaired in closure in response to drought. The results of further experiments
with ABA-insensitive Arabidopsis [Arabidopsis thaliana (L.)
Heynh.] mutants suggested that ABA contributes to resistance
against S. sclerotiorum through an antagonistic interaction with
oxalate. Melotto et al. (2006) investigated stomatal function
in Arabidopsis infected with Pseudomonas syringae pathovar
tomato (Okabe) Young, Dye & Wilkie. Mean stomatal aperture
was reduced 2 h after inoculation but returned to control levels after 4 h. It was suggested that the stomatal closure was an
innate immune response, triggered by bacterial pathogen-associated molecular pattern (PAMP) molecules, to prevent bacterial
entry through the stomatal pores. Experiments with a range of
Arabidopsis mutants and an NO synthase inhibitor indicated that
the stomatal closure was mechanistically linked to ABA signalling in guard cells. It was shown that the pathogen produced
the toxin coronatine to overcome the innate immune response
and effect stomatal reopening. Coronatine appeared to act downstream of ABA and downstream or independently of NO production. Liu et al. (2009) showed that the Arabidopsis protein
RIN4, a negative regulator of plant immunity, functions with
the plasma membrane H+-ATPases AHA1 and AHA2 to regulate
stomatal apertures, inhibiting the entry of bacterial pathogens
into the plant.
Gudesblat et al. (2009) found that Xanthomonas campestris
pathovar campestris (Pammel.) Dowson, as well as extracts from
pathogen culture supernatants, was able to reverse stomatal closure induced by bacteria or ABA in Arabidopsis. Pathogen rpfF
and rpfC mutant strains were incapable of reversing stomatal closure, indicating that suppression of stomatal response requires an
intact rpf/diffusible signal factor system. However, no specific
pathogen virulence factors that induced stomatal opening were
identified. Guard cell-specific Arabidopsis Mitogen-Activated
Protein Kinase3 (MPK3) antisense mutants failed to exhibit stomatal closure, indicating that MPK3 is required for the innate
immune response. In addition, ABA-induced stomatal closure
in MPK3 mutants was not affected by the pathogen, suggesting
that the virulence factor might target a component in the same
pathway as MPK3. Keinath et al. (2010) employed a reverse
genetics approach to study the contribution of plasma membrane
proteins in PAMP-triggered cellular responses in Arabidopsis.
Mutants of three candidates (DET3, AHA1, and FER) exhibited a
defect in the accumulation of reactive oxygen species (ROS), an
altered mitogen-activated protein kinase activation, and a defect
in PAMP-triggered stomatal closure. A further study (Mersmann
et al., 2010) highlighted a likely role for ethylene in PAMP-triggered stomatal closure. In the Arabidopsis ethylene-insensitive
mutants etr1 and ein2, accumulation of the bacterial PAMP
receptor FLS2 was diminished and stomatal closure as an early
immune response was abolished.
In interactions between peach (Prunus persica) and almond
[Prunus dulcis (Mill.) Webb] leaves with the necrotroph
Fusicoccum amygdale Delacr., the toxin fusicoccin acted
ahead of the fungus and triggered stomatal opening in the light
and in the dark (Turner and Graniti, 1976). Fusicoccin stimulates the plasma membrane H+-ATPase of guard cells, leading to the hyperpolarization of the plasma membrane, to an
increase of the K+in/H+out exchange (Marré, 1979), and to guard
cell turgor.
Induction of stomatal opening during oomycete
pathogenesis
A small number of studies have investigated the effects of
oomycete foliar pathogenesis on stomatal aperture. In contrast
to the effects reported for other pathogens, these studies have
all reported increases in stomatal conductance in green or chlorotic tissues irrespective of whether observations were made in
the light or in the dark (Farrell et al., 1969; Lindenthal et al.,
2005; Oerke et al., 2006). Allègre et al. (2007) studied stomatal
function in grapevine (V. vinifera) infected with Plasmopara
viticola (Berk & Curtis) Berl. & De Toni. Under progressive
water starvation, stomatal conductance of healthy leaves in
the light was reduced significantly, whereas conductance in
leaves of infected plants remained high. Stomata also remained
open in the dark in infected leaves. Cytological observations
indicated that stomatal opening was not related to mechanical
forces resulting from the presence of the pathogen in the substomatal cavity.
The results of these studies suggest that stomatal opening is a
common feature of oomycete pathogenesis, brought about by a
non-systemic compound produced by either the pathogen or the
infected plant. Oomycetes are known to produce disease effector
proteins which modulate plant defence and/or induce cell death
(Kamoun, 2006). It seems plausible that guard cell deregulation
is a mechanism that enables oomycetes to sporulate through
stomatal pores. As with non-oomycete pathogens, the failure of
stomata to close in the dark during oomycete pathogenesis has
implications for the ability of the affected crop to conserve water,
though the importance of night time transpiration effects remains
to be quantified.
Foliar pathogenesis and plant water relations | 4327
Host resistance and stomatal function
Several controlled environment studies have reported effects
on stomatal conductance following an incompatible interaction
between a resistant crop plant and a foliar pathogen. Impairment
of stomatal function in these pathosystems was associated with
hypersensitive cell death. McDonald and Cahill (1999) investigated stomatal aperture effects of Phytophthora sojae Kaufm.
& Gerd. foliar challenge on resistant and susceptible isolines of
soybean [Glycine max (L.) Merr.]. At a distance of up to 20 mm
from the infection site, stomata were closed 2 h post-inoculation
(hpi) in the resistant line, but remained open in the susceptible
line. This remained the case until 8 hpi, after which the stomata
in the resistant line gradually opened. It was considered by the
authors that the closure of stomata in the resistant line at a site
20 mm from the inoculation point indicated that a transmissible factor was involved in causing stomatal closure. Further
experiments showed that the factor was not induced by an abiotic elicitor or wounding, suggesting that it was a product of the
interaction of the pathogen with host cells that responded hypersensitively to the challenge.
Scharte et al. (2005) studied stomatal aperture and gasexchange effects of the incompatible interaction between
tobacco (Nicotiana tabacum L.) and Phytophthora nicotianae
Breda de Haan. The fraction of open stomata decreased strongly
at the infection site during the first 6 hpi but increased again at
the later stages of the challenge, while stomata in the uninoculated controls remained open throughout this period. The results
of gas-exchange analysis indicated that the stomatal closure was
associated with an inhibition of photosynthetic CO2 fixation. The
decline in photosynthesis affected a broader area of leaf tissue
than the stomatal closure at the inoculation site due to lateral gas
exchange within the intercellular space of the mesophyll. During
the first hour after challenge, the early accumulation of ROS was
accompanied by callose formation. These processes are typical
of the hypersensitive response (HR). The authors suggested that
stomatal closure could be initiated by ROS, as they are known to
activate calcium channels in guard cells and are involved in the
osmotic responses of stomata (e.g. Pei et al., 2000). It was also
thought that ROS as a signal for stomatal closure could explain
the localized response as callose restricts the diffusion of H2O2.
Prats et al. (2006) investigated stomatal aperture effects in
resistant and susceptible barley isolines following challenge with
B. graminis f.sp. hordei (Bgh), at an inoculum density of ~100
conidia mm–2. Mla resistance, based on HR, was compared with
mlo resistance which prevents penetration by papilla formation.
In the susceptible line Pallas, stomatal conductance of inoculated
leaves was decreased in the light compared with healthy leaves
during the 3 d following inoculation. Microscopic examination
showed that stomatal closure was associated with the presence of
a fungal colony in an adjacent cell. In the isoline P01 with Mla1
resistance, stomatal conductance in the light was initially lower
in inoculated leaves, but gradually increased after 3 d to levels
found in healthy leaves. Stomata of inoculated P01 leaves gradually lost their ability to close in the dark and remained locked
open from 3 days post-inoculation. Observation of locked-open
stomatal complexes indicated that HR had occurred in an adjacent cell or its immediate neighbour. Further experiments indi-
cated that locked-open stomata failed to close in response to
drought or to treatment with ABA.
Stomatal function of inoculated leaves of P22 and Riso-R,
both with mlo resistance, was less affected than for P01. In the
third dark period following inoculation, however, P22 leaves
failed to close fully in the dark. Cell death was more frequent in
P22 than in Riso-R, and was associated with stomatal malfunction. Penetration resistance was only associated with a transient
effect, and stomata recovered function after papilla deposition
was completed. A number of mechanisms which might cause this
transient effect (also present in Pallas and P01) were proposed,
including the generation of NO during papilla formation and the
export of protons from the substomatal apoplast to the guard cell
following contact with the fungal primary germ tube. Both of
these events are associated with stomatal closure. A mechanism
was also proposed for the inability of stomata to close in the
dark when in proximity to epidermal cells that have died due to
HR (e.g. as in P01) or as a consequence of uncontrolled H2O2
production (e.g. as in P22). It was suggested that the collapse of
epidermal cells could reduce their turgor relative to the stomatal
complex and perhaps reduce water flow to the subsidiary cell
which would in turn lose turgor. Opening of the stomatal aperture depends on the balance between guard cell and epidermal
subsidiary cell turgor.
In further work, these authors investigated the effects of
reduced Bgh inoculum densities on stomatal function in P01 and
P02 which carry the Mla1 and Mla3 resistance genes, respectively (Prats et al., 2010). It was found that, in general, impairment of stomatal function in the light and dark was positively
correlated with inoculum density from 1 to 100 conidia mm–2.
Even at the lowest inoculum density, significant differences from
healthy controls were observed after a number of diurnal cycles.
There was a delayed stomatal response in the dark in P02 compared with P01, and this was associated with a delayed HR. The
fact that lower inoculum densities have been shown to affect stomatal conductance suggests that inoculum densities found in the
field may be sufficient to affect stomatal function.
Prats et al. (2007) investigated stomatal responses of susceptible and resistant barley and wheat to challenge by the biotrophic
brown rust fungus (Puccinia hordei Otth and P. triticina Erikss.
respectively). The susceptible barley cultivar (cv.) ‘Gold’ was
compared with cv. ‘Estate’ carrying resistance gene Rph3 associated with a chlorotic reaction and cv. ‘Cebeda Capa’ carrying resistance gene Rph7, associated with a necrotic reaction.
For all three cultivars, the challenge resulted in large reductions
in stomatal conductance in the light but little effect in the dark
compared with healthy controls. The susceptible wheat line
Thatcher was compared with lines carrying Lr24 associated with
a mainly chlorotic reaction, and Lr20 associated with chlorotic
and necrotic symptoms. For all three lines, there was little effect
on stomatal conductance in the dark compared with healthy
controls. In the light, there were reductions in the order Lr20
> Thatcher > Lr24, indicating that Lr24 resistance was likely
to have a lesser impact on photosynthesis than resistance based
on Lr20.
Although the studies above were conducted in controlled
environments, they show clear, consistent effects that could
plausibly translate to field conditions and contribute to the yield
4328 | Grimmer et al.
penalty thought to be associated with some resistance genes
(Brown, 2002). These findings have important implications for
the deployment of resistance in crop cultivars and suggest that
microphenotyping (microscopic examination of host tissue cell
death traits) should be used to characterize resistance genes
before they are used in breeding programmes. The yield penalty
evoked within a cultivar is likely to be amplified according to
the number of different resistance genes that are combined to
produce commercially acceptable resistance against all the economically important pathogens. As well as constraints on photosynthesis imposed by resistance expression, possible effects of
lock-open on WUE should be determined.
Non-host resistance and stomatal function
There have been a limited number of studies on the effects on stomatal conductance of foliar challenge with a non-host pathogen.
Melotto et al. (2006) challenged Arabidopsis leaves with the nonhost human pathogen Escherichia coli (Migula) Castell. & Chalm.
O157:H7 and found that the average width of the stomatal aperture was decreased by ~3-fold at 2, 4, and 8 hpi. It was suggested
that this was an innate immune response to conserved, bacterial
PAMP molecules. Stomatal closure was effectively prevented by
an inhibitor of NO synthase, indicating that NO was required.
Shafiei et al. (2007) found that foliar challenge of Arabidopsis
with the wheat rust pathogen P. triticina resulted in a reduction in
the percentage of open stomata compared with mock-inoculated
controls from 3 hpi. Pathogen-induced stomatal closure was limited in the Arabidopsis line aba3 compromised in the biosynthesis
of ABA, indicating that it was an ABA-dependent process.
Incompatible interactions
Compatible interactions
Impairment of stomatal opening
Fungi &
Viruses
Prats et al. (2010) investigated the effects of foliar challenge
of barley with the non-host oat pathogen B. graminis f.sp. avenae. Stomatal opening in the light and closing in the dark were
both impaired, and this was associated with the death of adjacent
epidermal cells. There were no differences in cell death or stomatal conductance responses between lines varying for resistance to the host pathogen B. graminis f.sp. hordei, indicating that
the race-specific resistance genes had no influence on the degree
of stomatal dysfunction.
Studies on stomatal responses to non-host pathogen challenge
have necessarily taken place in a controlled environment in order
to preclude challenge from other host and non-host organisms.
However, the results have important implications for field crops.
There are a wide range of non-host pathogens in the crop phyllosphere; if all or some of these disrupt stomatal conductance then
it suggests that their presence has a discernible, detrimental effect
on photosynthesis and therefore yield. The magnitude of this effect
could conceivably be reduced through selection for cultivars that
maintain stomatal function following non-host pathogen challenge.
Conclusions
WUE is likely to become an increasingly important measure of
the sustainability of crop production. The effects of plant pathogens on WUE have not been considered adequately in this context. A range of foliar pathogens have been shown to disrupt the
normal stomatal regulation of transpiration and gas exchange
(summarized in Fig. 1). Deleterious effects on WUEI have been
demonstrated in a number of pathosystems. Impairment of sto-
Impairment of stomatal closing
Bacteria
Host innate
immune response
Bacterial
coronatine
Oomycete effectors
Oomycetes
Fungal
biotrophs &
Oomycetes
Non-host
Fungal
biotrophs &
Bacteria
Necrotroph effectors
e.g. oxalate, fusicoccin
Fungal
necrotrophs &
Oomycetes
Powdery mildew
Non-host powdery
mildew
Figure 1 Schematic diagram representing effects of various plant–pathogen interactions on stomatal function.
Foliar pathogenesis and plant water relations | 4329
matal opening in the light has implications for the ability of the
affected plant to assimilate CO2 for photosynthesis. Cuticular breach by pathogen structures, and impairment of stomatal
closing in the dark have implications for the ability to conserve
water. The reports that susceptible and resistant plant–pathogen
interactions can inhibit the normal ABA-mediated stomatal closure response of plants to drought stress suggest the potential
for pathogen challenge to impact on crop performance under
drought environments. With more frequent summer droughts
predicted by climate change models (Jenkins et al., 2009), yield
losses to drought are likely to be exacerbated. Optimizing disease resistance genes in new cultivars will be one way of combating these drought effects. In addition, improving WUE will
increase the amount of water returned to the hydrological system
for reuse, leading to conservation of water resources for use in
irrigating other crops, and increased water flows in rivers, water
levels in wetland areas, and aquifer recharge. A major challenge
for researchers should be to test whether deleterious effects on
growth and WUE can be detected in the field, where rainfall,
evaporation, and competition between plants also exert effects.
There are a number of reports on the molecular mechanisms
by which pathogen- and host-derived compounds affect the normal regulation of stomata following foliar pathogen attack (e.g.
Scharte et al., 2005; Melotto et al., 2006; Shafiei et al., 2007).
A second challenge for researchers should be to link molecular
physiology to leaf and whole-plant physiology. Many of the tools
needed to achieve this are already available, such as: (i) intricate
cellular dissection techniques; (ii) segregating populations and
near-isogenic lines which vary for key disease resistance genes;
(iii) Arabidopsis mutants in genes which control stomatal movements; and (iv) synthesized compounds based on plant hormones
and pathogen toxins/elicitors which affect guard cell function.
Ayres PG, Zadoks JC. 1979. Combined effects of powdery mildew
disease and soil water level on the water relations and growth of
barley. Physiological Plant Pathology 14, 347–361.
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