Acute Physiological Responses to High-Intensity Resistance ...
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Physiological responses of Physiological responses of BurkholderiaBurkholderia phytofirmansphytofirmans strain strain PsJNPsJN
colonized plantlets of grapevine (colonized plantlets of grapevine (VitisVitis viniferavinifera L.) to L.) to low nonlow non--freezing temperatures freezing temperatures
Andreas I. THEOCHARIS
Laboratoire de Stress, Défenses et
Reproduction des Plantes
Unité de Recherche Vignes et Vins de Champagne -
Stress et Environnement UFR Sciences Exactes et Naturelles
A thesis submitted for the Degree of Doctor of Philosophy
culturally appropriate
socially just
economicallyviableholistic
scientific approach
ecologicallysound
An agriculture is Sustainable
Sustainable agriculture
(ATTRA, 2003;2005)
Beneficial microorganisms
Sustainable development
General introduction
The idea of a world where people protect the environment as they carry out their day-to-day activities
Literature Review
(Bakkeret al. 2003; Dobbelaereet al. 2003; Compantet al., 2005)
Help in management ofenvironnemental Stress
Abiotic stress
Biotic stress
Directly
•Induced systemic resistance (ISR)
Indirectly
Plant growth promotion
Plant growth promoting rhizobacteria (PGPR) & plants
The beneficial effects of PGPR
•ACC deaminaseactivity
• Nitrogen fixation
• Solubilization of minerals• etc
•Synthesis of anti-fungal compounds
•Synthesis of fungal cell wall-lysing enzymes•etc
Literature Review
ISR & signaling pathways
(Pieterseet al., 1998 ;Vallad & Goodman, 2004)De Meyer et al., 1999; Timmusk &
Wagner, 1999; Park & Kloepper, 2000 ; Magnin-Robert et al. 2007
ISR
SA-, JA-, and ET
PR genes
PRproteins
NPR1
A state of increased defensive capacity developed in
by
through activation of latent resistance mechanisms
ISR-inducing PGPR
Literature Review
(Ryalset al., 1996; Doke, 1996; Sticheret al., 1997; Conrathet al., 2002; 2006; Goellner & Conrath, 2008)
Systemic resistance by primed physiological responses
The phenomenon of priming
has been associated with
a quicker and/or stronger activation
of plant cellular defenses upon exposure to environmental stress
Priming
Systemic response induced by or
colonization of plant roots by ISR-inducing PGPR
Pathogens
Literature Review
Priming mechanism : Causes and Effects
Pathogens,PGPRs
SAR-inducers
Cold acclimationa) Primed physiological stage
PrimingOsmotic stress,
Wounding
Temperature stressElicitors
Pathogens
b) Challenge with stress
Stronger responses against stress
Faster responses against stress
(Conrath et al., 2002; 2006; Goellner & Conrath, 2008)
c) Potentiated physiological reaction of plants
Literature Review
Strain PsJN is able to establish rhizosphere and endophytic populations in plants
Burkholderia phytofirmans strain PsJN
(Pillay & Nowak, 1997; Bensalimet al., 1998; Sharma & Nowak, 1998; Compantet al., 2005)
A member of the genus Burkholderia belongs to the ß-Proteobacteria
• better water management in plants• increased resistance to temperature stress
• increased resistance against pathogens
Plant growth stimulation
•phenylalanine ammonia lyase and phenolics
induction of defense mechanisms&
• ACC deaminase activity
• a characteristic larger root system
• sturdier stems & greater lignin deposition
•increased levels of chlorophyll & cytokinins
Literature Review
Fruitingcutting
Low non-freezing temperature
(after 2 weeks)
Promoted growth and physiological
activity
Enhanced level of proline, total phenolic s &
starch deposition
Improved photosynthetic
capacity
(Ait Barka et al, 2006)
Literature Review
Grapevine –B. phytofirmans strain PsJNinteraction
Significant capacity of bacterized plantlets to withstand chilling
(Compantet al., 2005, 2008)
Rhizosphere & endophytic population in grapevine roots, vegetative and
reproductive organsColonization of
strain PsJN
resistance toBotrytis cinerea
Faster growth & development with robust root system
Beneficial effects of strain PsJN in
grapevine plantlets
(Ait Barka et al, 2000, 2002)
in vitro-plants
4°C
Tropical &subtropical origin
Not able to survive in low non-freezing
temperatures(Chilling stress)
( Lyons, 1973; Raison & Lyons, 1986; Wang, 1990; Tomashow, 1999 )
Literature review
Plant sensitivity in low non-freezing temperatures(0°C - 15°C )
Plants
The biology of plantto cold
Temperate origin (e.g. grapevine)
Cold acclimation
Adaptation to low non-freezing temperatures
Freezing tolerance (< 0°C)
Literature review
(Raison & Lyons, 1970; Lyons, 1973)
Liquid-Crystalline
phase
transitionSolid gel
Solute leakage & disrupted ion
balance
Injury &death of cells and tissues
Increased permeability
Increased activation of energy-bound
enzymes
Cessation of protoplasmic
streaming
Imbalance in metabolism
Accumulation of toxic metabolites
e.g. acetaldehydes, ethanol etc
Reduced ATP supply
Return to normal
metabolism
Brief exposure and return to 20 °C
Chilling injury in sensitive plants
Prolonged exposure
Cell membranes
Literature Review
Plant biologyin cold
AOS accumulation induces chilling injuries in sensitive plants
Inactivation of enzymes
Lipid peroxidation
Protein degradation
( Raison & Lyons, 1986; McKersie & Lesham, 1994; Asada & Takahashi, 1987; Peiet al., 2000)
PS I, II
Plasma membranes
Chilling
AOS
Damage to DNA
Sensitive plants
Detrimental the prolonged
presence of high levels of AOS
membrane rigidification & cell
death
Production ofaldehydes &
malonedialdehydes
degradation of the polyunsaturated fatty
acids
( Uemura & Steponkus, 1999; Thomashow, 1999; Xin & Browse, 2000; Chang et al., 2001; Browse & Xin, 2001)
Responses of insensitive plants to low non-freezing temperatures
Accumulation of[Ca2+
cyt]
Accumulation of AOS and activation of scavenge system
Change in gene expression and protein synthesis
Accumulation of sugarsand prolines
Modification in plantmembranes
Acclimation to cold
Biochemical & physiological
changes
Elicitation of stable developmental
responses to low temperatures
Literature Review
Photosynthetic acclimation
a regulatory network of
genes
Induction of cold related
genes (COR ) Cold acclimation
Induction ofantioxidant
systemsAccumulation of
antioxidantenzymes
(Tähtiharjuet al. 1997; Peiet al., 2000; Thomashow, 2001; Fowler & Thomashow, 2002 )
The process of cold acclimation & the signal transduction pathways
H2O2
Cold acclimation
Adaptation to low temperatures
Biochemical andphysiological
changes
AtCBF2
AtCBF3
AtCBF4
AtCBF1
Transcription factors
CBF
[Ca2+cyt]
Accumulationcoffree proline and total
soluble sugars
Signaling molecule
Up-regulation of COR gene products
Freezing tolerance
Literature Review
Literature Review
Plant biologyin cold
Cold Stress
Nutritional role during acclimation
Stabilizerof membrane
Regulators of some enzymatic systems Scavengers of reactive
oxygen species
Inducer of stress-related genes
accumulation of carbohydrates
cryoprotectantsFree prolineaccumulation
Reduction of plant activegrowth
Decreased demand forthe products ofphotosynthesis
Enhanced activitiesof Calvin cycle
enzymes
(Sasaki et al., 1996; Ögrenet al., 1997; Dörfflinget al., 1997 ; Greer et al., 2000)
Accumulation of cryoprotectant contents in acclimated plants
Insensitive plants ?
How does grapevine sense the root colonization by bacteria and what are
the molecular and physiological changes that occur in grapevine by this
interaction?
Which grapevine defense mechanisms can be activated by these changes
and how could they help grapevine to better tolerate “cool” climate?
The three objectives of the project
Despite the available information by previous studies,
several questions remained regarding the beneficial
interaction between
&
Study of response of grapevine plantlets after root inoculation by Bulkholderia phytofirmans strain
PsJN
Objective I
Characterization of defense mechanisms activated in fully bacterized plantlets upon exposure to low non-freezing
temperatures
Objective II
Objective III
cDNA-AFLP differential gene expression analysis of physiological state induced by Burkholderia phytofirmans strain PsJN in grapevine upon low non-
freezing temperatures
The three objectives of the project
Study of response of grapevine plantlets after root inoculation by Bulkholderia phytofirmans strain
PsJN
Objective I
goalInvestigation whether PsJN strain is able to stimulate the defense
mechanism by induction of selected defense genes after root inoculation,
similarly to ISR-type responses.
Objective I
May this stimulation promote the plant resistance against cold?
Changes in pattern of defense gene expression in leaves by qRT-PCR
Objective IVitis vinifera L. cv. Chardonnay clone 7573 16-h fluorescent light at 26°C
micro-cuttings1 nodal explant
propagation
2 ml of inoculum ofstrain PsJN
6wk-old plantlets
+
Strain PsJN(3 x 108 CFU\ml of
inoculum)
two loops of strain PsJN
King’s B liquidmedium
immersion
Plant bacterizationprocess
re-suspenedin PBS
122448
Hours after root inoculation
Experiments were repeated twice
0
5
10
15
20
0 12 24 36 48
Ind
uction
Hours post-inoculation
Phenylalanine ammonia-lyase (VvPAL)
0
10
20
30
40
0 12 24 36 48
Ind
uctio
n
Hours post-inoculation
Stilbene synthase (VvStSy)
0
2
4
6
8
0 12 24 36 48
Ind
uct
ion
Hours post-inoculation
Lipoxygenase (VvLOX)
Objective I
Genes encoding enzymes from phenylopropanoid& octadecanoid pathways
0
2
4
6
8
10
0 12 24 36 48
Ind
uctio
n
Hours post-inoculation
Chitinase 4c (VvChit4c)
02468
1012141618
0 12 24 36 48
Ind
uctio
n
Hours post-inoculation
Glucanase (VvGluc)
0
50
100
150
200
0 12 24 36 48
Ind
uction
Hours post-inoculation
Protease inhibitor (VvPIN)
Objective I
Genes encoding for pathogenesis-related proteins (PRs)
Objective I
ii) Induction of systemic responses by strain PsJN
i) Induction of defense mechanism after root inoculation
•Analysis of the impact of the strain PsJN on the chilling tolerance under ISR condition
•Analysis of SA and JA levels in plants to discriminate the pathway(s) involved in the establishment of ISR
Future work for characterization of defense signal as ISR:
ISR?
PRs
Conclusion &Discussion
iii) The induction of transcript accumulation involves genes encoding for PRs, similarly to other ISR-inducing PGPR, suggesting an overlapping between ISR and SAR
1st
goal
Study of the expression pattern of well-characterized
grapevine defence genes and CBF transcription factors in
grapevine plantlet leaves
Objective II (1st goal)
Objective IICharacterization of defense mechanisms activated in fully bacterized plantlets upon exposure to low non-
freezing temperatures
strain PsJN
(3 x 108
CFU\ml)
2wk-old
plantlets
+
CBF4 transcription factors
9 h24 h
Changes in pattern of VvStSy, VvPAL, VvLOX, VvGluc, VvChit4candVvChit1b expression in leaves
Time after cold treatment
6wk-old fully bacterized
plantlets
after 4 wk
Vitis vinifera L. cv. Chardonnay clone 7573
16-h light/ 8-h dark at
26°C micro-cuttings1 node
propagationStrain PsJN
Plant bacterizationprocess
24 h48 h72 h2wk
10°C/ 4°C , 16 h light/ 8 h dark
Objective II
1st
goal
Experiments were repeated 3 times
Objective II
non-bacterized 26°C
non-bacterized 4°C
bacterized 26°C
bacterized 4°C
Time after cold treatment
Time after cold treatment
1st
goal
Genes coding enzymes from phenylopropanoid pathways
Objective II
Time after cold treatment
Time after cold treatment
non-bacterized 26°C
non-bacterized 4°C
bacterized 26°C
bacterized 4°C
Time after cold treatment
1st
goal
Genes encoding for pathogenesis-related proteins (PRs)
Objective II
non-bacterized 26°C
non-bacterized 4°C
bacterized 26°C
bacterized 4°C
1st
goal
Gene encoding enzymes from octadecanoid pathway & CBF4 transcription factor
0
20
40
60
80
100
120
140
160
180
200
9 h 24 h
Ind
uct
ion
Time after cold treatment
CBF4
Objective II
1st
goal
Conclusion &Discussion
1. In grapevine plantlets, low temperatures induced the defense-related gene
transcripts & cold specific transcription factor CBF4 according to the
phenomenon of priming
2. From analysed genes, except for CBF4, chitinases and glucanasesare of
special interest since they exhibit both antifreeze and antifungal activities
4. This induction of grapevine defense mechanism may be correlated with
previous results showing that leaf cells of bacterized plantlets are less
affected by cold, and it further indicates that B. phytofirmans strain PsJN
may improve grapevine resistance to low non-freezing temperatures (Ait
Barkaet al., 2006)
3. The clear potentiated expression of LOX in bacterized plantlets after cold
stress suggests that JA signal transduction pathway could be involved in the
process of cold acclimation induced by B. phytofirmans.
Objective IICharacterization of defense mechanisms activated in fully bacterized plantlets upon exposure to low non-
freezing temperatures
Determination of proline accumulation and analysis of changes in the level
of lipid peroxidation markers (aldehydes, malondialdehydes) and hydrogen
peroxide
2nd
goal
Objective II (2nd goal)
a
b
c
a
b
a
c
a
b
a
c
a
b
a
c
a
b
a
c
a
Objective II
aa
bb
aa
b b
a a
b b
ab
c
d
ab
c
d
non-bacterized 26°C
non-bacterized 4°C
bacterized 26°C
bacterized 4°C
2nd
goal
Analysis of stress markers (Prolines & Hydrogen peroxide)
Objective II
non-bacterized 26°C
non-bacterized 4°C
bacterized 26°C
bacterized 4°C
ab
a
c
a
b
a
b
a
b
a
b
a
b
a
c
a
b
a
c
a
b
a
c
a
b
a
c
a
b
a a
b
a
c
b
a
c
a
b
2nd
goal
Analysis of lipid peroxidation markers (Aldehydes & MDA)
Objective II
2nd
goal
Conclusion &Discussion
3. B. phytofirmans provokes stronger H2O2 accumulation within the first 3 days of
treatment but also speeds up the decrease of H2O2 level after 1 week. Probably, H2O2
triggers the synthesis of antioxidant enzymes such as catalaseor peroxidase that
scavenge ROS and help the plant to overcome cold conditions
2. Proline is the most well characterized stress responsive molecule, and it is not
surprizing that in grapevine, accumulation of proline appeared as a response to cold
acclimation process (Ait Barkaet al., 2006). The accumulation of proline in bacterized
plantlets according to the phenomenon of priming, reveals the protective role of
bacteria
1. Apart from gene expression, plant responds to coldness by stress-related
metabolites such as proline, hydrogen peroxide or aldehydes & malondialdehydes, in
similar way to priming phenomenon
4. Finally, aldehydes and MDA are accumulated by almost similar ways to those
reported for H2O2, confirming that B. phytofirmans speeds up grapevine reaction to
cold shift and later favours the acclimation process to cold temperatures, showing that
the presence of PsJN improves the loss of permeability of membranes as response to
cold (Barkaet al. 2006)
Objective IICharacterization of defense mechanisms activated in fully bacterized plantlets upon exposure to low non-
freezing temperatures
Determination of starch deposition and soluble sugar
(total soluble sugars, sucrose, glucose, fructose) accumulation in grapevine
plantlet leaves upon exposure to low non-freezing temperatures
3rdgoal
Objective II (3rd goal)
Objective II
b
a
ba
c
a
ba
c
a
ba
c
a
cb
a
b
cb
ab c
d
ab c
d
a
b
c
d
a
b
c
d
a
b
c
d
non-bacterized 26°C
non-bacterized 4°C
bacterized 26°C
bacterized 4°C
3rdgoal
Starch deposition & total soluble sugars accumulation
Objective II
non-bacterized 26°C
non-bacterized 4°C
bacterized 26°C
bacterized 4°Ca
ba
c
a
b
a
c
a
b
a
c
a
b
a
c
a
b
a
c
a
ba
c
a
b
a
c
a
b
a
c
a
b
aa
c
b
dc
a
b
a
c
a
b
a
c
a
b
a
c
a
b
a
c
a
b
c
b
3rdgoal
Enzymatic analysis of soluble sugars
Objective I
3rdgoal
1. According to our results strain PsJN affects carbohydrate metabolism
in grapevine plantlets in normal growth conditions related with the
stimulation of net photosynthesis (Ait Barkaet al., 2006), which may
contribute to sugar accumulation
3. By higher accumulation of carbohydrates in bacterized plantlets, we
could address that PsJN is a PGPR that primes several physiological
responses of grapevine plantlets under cold stress including the accumulation
of soluble sugars and starch, speeding up the process of cold acclimation
2. Cold acclimation induces an increase of both soluble sugars and starch
in grapevine grown in the vineyard (Ait Barka & Audran, 1996) or in our
plantlets grown in growth chamber, oppositely with starch that may be
converted into soluble saccharides during cold exposure in some species
B. Phytofirmans is an ISR-type PGPR able to potentiatethe physiological response of cold acclimation and to prime the grapevine development and growth upon low non-
freezing temperatures
B. Phytofirmans is an ISR-type PGPR able to
prime
the induction of known defense genes & genes with specific
role in cold acclimation process
StSyPAL
Chit4c
Chit1b
GlucLOX
CBF4
sucrose
the accumulation of cryoprotectant
contents
prolinetotal soluble
sugars
starch
fructoseglucose
the faster degradation of lipid peroxidation and
stress markers
malondialdehyde
hydrogen peroxide
aldehydes
General conclusions
General conclusions
Future prospects
Prospect I
Investigation of primed- physiological state of V. vinifera L. induced
by Burkholderia phytofirmans strain PsJN by transcription analysis.
The identification and analysis of gene expression profile may support
our knowledge about the signalling pathways of priming phenomenon
Prospect II
Using the molecular tools, like specific mutants of A. thaliana, for
analysis of signaling pathways induced byBurkholderia
phytofirmans strain PsJNn and for better understanding of
beneficial effects in plants
Greek Scholarship Foundation
IKY AcknowledgmentsAcknowledgments