1. Unité de Recherche Vignes et Vins de
Champagne Stress et Environnement
UFR Sciences Exactes et Naturelles
Laboratoire
de Stress, Défenses et
Reproduction des Plantes
Physiological responses of
Burkholderia phytofirmans strain PsJN
colonized plantlets of grapevine (Vitis vinifera L.) to
low non-freezing temperatures
Andreas I. THEOCHARIS
A thesis submitted for the Degree of Doctor of Philosophy

2. Sustainable development
Literature
Review
The idea of a world where people protect the environment
as they carry out their day-to-day activities
culturally
appropriate
An agriculture
is Sustainable
socially just
Sustainable
General introduction
ecologically agriculture
sound
economically
holistic
viable
scientific
approach
(ATTRA, 2003;2005)
Beneficial microorganisms

3. Plant growth promoting rhizobacteria (PGPR) & plants
Literature
Review
Plant growth promotion
• Nitrogen fixation
• Solubilization of minerals
• etc
Help in management of
environnemental Stress
Abiotic stress
The beneficial effects of PGPR
Biotic stress
•ACC deaminase
activity
Directly
•Synthesis of anti-fungal compounds
•Synthesis of fungal cell wall-lysing enzymes
•etc
Indirectly
•Induced systemic resistance
(ISR)
(Bakker et al. 2003; Dobbelaere et al. 2003; Compant et al., 2005)

4. ISR & signaling pathways
Literature
Review
A state of increased defensive capacity developed in
ISR-
by inducing PGPR
through activation of latent resistance mechanisms
PR
proteins
NPR1
PR
genes
SA-, JA-,
and ET
ISR
(Pieterse et al., 1998 ;Vallad & Goodman, 2004)
De Meyer et al., 1999; Timmusk &
Wagner, 1999; Park & Kloepper, 2000 ;
Magnin-Robert et al. 2007

5. Systemic resistance by primed physiological responses
Literature
Review
Systemic response induced by
Pathogens
colonization of plant roots by
or
ISRinducing PGPR
The phenomenon of priming
Priming
has been associated with
a quicker and/or stronger activation
of plant cellular defenses upon exposure to environmental stress
(Ryals et al., 1996; Doke, 1996; Sticher et al., 1997; Conrath et al., 2002; 2006; Goellner & Conrath, 2008)

6. Priming mechanism : Causes and Effects
Literature
Review
a) Primed physiological stage
Cold acclimation
Pathogens,
PGPRs
b) Challenge with stress
Elicitors
SAR-inducers
Osmotic stress,
Priming
Pathogens
Temperature stress
Wounding
c) Potentiated physiological
reaction of plants
Faster responses against
stress
Stronger responses against
stress
(Conrath et al., 2002; 2006; Goellner & Conrath, 2008)

7. Burkholderia phytofirmans strain PsJN
Literature
Review
A member of the genus Burkholderia belongs to the ß-Proteobacteria
Strain PsJN is able to establish rhizosphere and endophytic populations in plants
Plant growth stimulation
• a characteristic larger root system
• sturdier stems & greater lignin deposition
•increased levels of chlorophyll & cytokinins
&
induction of defense mechanisms
•phenylalanine ammonia lyase and phenolics
• ACC deaminase activity
• better water management in plants
• increased resistance to temperature stress
• increased resistance against pathogens
(Pillay & Nowak, 1997; Bensalim et al., 1998; Sharma & Nowak, 1998; Compant et al., 2005)

8. Grapevine – B. phytofirmans strain PsJN interaction
Literature
Review
Significant capacity of bacterized plantlets to
withstand chilling
in vitro-plants
Promoted growth
and physiological
activity
Beneficial effects
of strain PsJN in
grapevine
plantlets
Faster growth & development
with robust root system
resistance to
Botrytis cinerea
Fruiting
cutting
Colonization of
strain PsJN
Low non-freezing temperature
(after 2 weeks)
(Ait Barka et al, 2006)
4°C
Rhizosphere & endophytic population
in grapevine roots, vegetative and
reproductive organs
Enhanced level of
proline, total phenolic s &
starch deposition
(Ait Barka et al, 2000, 2002)
Improved
photosynthetic
capacity
(Compant et al., 2005, 2008)

9. Plant sensitivity in low non-freezing temperatures
(0°C - 15°C )
Literature
review
Plants
Tropical &
subtropical origin
Not able to survive in
low non-freezing
temperatures
(Chilling stress)
The biology of plant
to cold
Temperate origin
(e.g. grapevine)
Cold
acclimation
Adaptation to low
non-freezing
temperatures
Freezing
tolerance (< 0°C)
( Lyons, 1973; Raison & Lyons, 1986; Wang, 1990; Tomashow, 1999 )

10. Chilling injury in sensitive plants
Literature
review
Cell membranes
phase
LiquidSolid gel
Crystalline
transition
Return to
normal
metabolism
Brief exposure and
return to 20 °C
Increased
permeability
Increased
activation of
energy-bound
enzymes
Cessation of
protoplasmic
streaming
Reduced
ATP supply
Imbalance in
metabolism
Solute leakage &
disrupted ion
balance
Accumulation of
toxic metabolites
e.g. acetaldehydes,
ethanol etc
Prolonged
exposure
Injury &death of cells
and tissues
(Raison & Lyons, 1970; Lyons, 1973)

11. AOS accumulation induces chilling injuries in sensitive plants
Literature
Review
Plant biology
in cold
Sensitive
plants
Plasma
membranes
Chilling
Inactivation of
enzymes
PS I, II
Damage to DNA
Detrimental the
prolonged
presence of high
levels of AOS
AOS
Protein
degradation
Lipid
peroxidation
membrane
rigidification & cell
death
Production of
aldehydes &
malonedialdehydes
degradation of the
polyunsaturated fatty
acids
( Raison & Lyons, 1986; McKersie & Lesham, 1994; Asada & Takahashi, 1987; Pei et al., 2000)

12. Responses of insensitive plants to low non-freezing temperatures
Literature
Review
Accumulation of sugars
and prolines
Modification in plant
membranes
Accumulation of AOS and
activation of scavenge system
Biochemical &
Acclimation
physiological
to cold
changes
Accumulation of
[Ca2+cyt]
Photosynthetic
acclimation
Change in gene expression
and protein synthesis
Elicitation of stable
developmental
responses to low
temperatures
( Uemura & Steponkus, 1999; Thomashow, 1999; Xin & Browse, 2000; Chang et al., 2001; Browse & Xin, 2001)

13. The process of cold acclimation & the signal
transduction pathways
Literature
Review
Induction of
antioxidant
systems
Cold acclimation
Signaling
molecule
Transcription
factors
AtCBF1
H2O2
[Ca2+cyt]
Up-regulation of
COR gene products
Biochemical and
physiological
changes
Accumulationcof
free proline and total
soluble sugars
Accumulation of
antioxidant
enzymes
CBF
a regulatory
network of
genes
Induction of
cold related
genes (COR )
AtCBF2
AtCBF3
AtCBF4
Cold
acclimation
Adaptation to
low temperatures Freezing
tolerance
(Tähtiharju et al. 1997; Pei et al., 2000; Thomashow, 2001; Fowler & Thomashow, 2002 )

14. Accumulation of cryoprotectant contents in acclimated plants
Literature
Review
Plant biology
in cold
Insensitive
plants
Cold Stress
?
Free proline
accumulation
Reduction of plant active
growth
Enhanced activities
Decreased demand for
of Calvin cycle
the products of
enzymes
photosynthesis
cryoprotectants
accumulation of
carbohydrates
Inducer of stressStabilizer
related genes
of membrane
Regulators of some
Scavengers of reactive
enzymatic systems
oxygen species
Nutritional role
during acclimation
(Sasaki et al., 1996; Ögren et al., 1997; Dörffling et al., 1997 ; Greer et al., 2000)

15. Despite the available information by previous studies,
several questions remained regarding the beneficial
interaction between
The three objectives of the project
&
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?

16. The three objectives of the project
Objective I
Objective II
Study of response of grapevine plantlets after root
inoculation by Bulkholderia phytofirmans strain
PsJN
Characterization of defense mechanisms activated in fully
bacterized plantlets upon exposure to low non-freezing
temperatures
cDNA-AFLP differential gene expression analysis of physiological state
Objective IIIinduced by Burkholderia phytofirmans strain PsJN in grapevine upon low nonfreezing temperatures

17. Objective I
Study of response of grapevine plantlets after root
inoculation by Bulkholderia phytofirmans strain
PsJN
Objective I
Investigation whether PsJN strain is able to stimulate the defense
goal
mechanism by induction of selected defense genes after root inoculation,
similarly to ISR-type responses.
May this stimulation promote the plant resistance against cold?

18. Vitis vinifera L. cv. Chardonnay clone 7573
16-h fluorescent light at 26°C
Objective I
micro-cuttings
1 nodal explant
two loops of
strain PsJN
propagation
King’s B liquid
medium
re-suspened
in PBS
Strain PsJN
(3 x 108 CFUml of
inoculum)
Changes in pattern of defense gene
expression in leaves by qRT-PCR
Plant bacterization
process
12
24
48
Hours after root
inoculation
+
immersion
2 ml of inoculum of
strain PsJN
6wk-old plantlets
Experiments were repeated twice

19. Genes encoding enzymes from phenylopropanoid
& octadecanoid pathways
Objective I
Stilbene synthase (VvStSy)
Induction
40
20
15
10
5
0
30
20
10
0
0
12
24
36
48
0
12
24
Lipoxygenase (VvLOX)
8
6
4
2
0
0
36
Hours post-inoculation
Hours post-inoculation
Induction
Induction
Phenylalanine ammonia-lyase
(VvPAL)
12
24
36
Hours post-inoculation
48
48

20. Genes encoding for pathogenesis-related proteins (PRs)
Objective I
Induction
Chitinase 4c (VvChit4c)
10
8
6
4
2
0
0
12
24
36
48
Hours post-inoculation
Protease inhibitor (VvPIN)
18
16
14
12
10
8
6
4
2
0
Induction
Induction
Glucanase (VvGluc)
0
12
24
36
Hours post-inoculation
48
200
150
100
50
0
0
12
24
36
Hours post-inoculation
48

21. i)
Induction of defense mechanism after root inoculation
Objective I
PRs
ii) Induction of systemic responses by strain PsJN
Conclusion &
Discussion
ISR?
iii) The induction of transcript accumulation involves genes encoding
for PRs, similarly to other ISR-inducing PGPR, suggesting an
overlapping between ISR and SAR
Future work for characterization of defense signal as ISR:
•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

22. Objective II
Characterization of defense mechanisms activated in
fully bacterized plantlets upon exposure to low nonfreezing temperatures
Objective II (1st goal)
1st
goal
Study of the expression pattern of well-characterized
grapevine defence genes and CBF transcription factors in
grapevine plantlet leaves

23. Vitis vinifera L. cv. Chardonnay clone 7573
16-h light/
8-h dark at
26°C
microcuttings
1 node
Objective II
propagation
Strain PsJN
1st
goal
CBF4 transcription
factors
Plant bacterization
process
9h
24 h
Changes in pattern of VvStSy,
VvPAL, VvLOX, VvGluc, VvChit4c
and VvChit1b expression in leaves
24 h
48 h
72 h
Time after
cold treatment
2wk
10°C/ 4°C ,
16 h light/ 8 h dark
after 4 wk
+
strain PsJN
(3 x 108
CFUml)
2wk-old
plantlets
6wk-old fully bacterized
plantlets
Experiments were repeated 3 times

24. Genes coding enzymes from phenylopropanoid pathways
Objective II
1st
goal
non-bacterized 26°C
bacterized 26°C
non-bacterized 4°C
bacterized 4°C
Time after cold treatment
Time after cold treatment

25. Genes encoding for pathogenesis-related proteins (PRs)
Objective II
1st
goal
non-bacterized 26°C
bacterized 26°C
non-bacterized 4°C
bacterized 4°C
Time after cold treatment
Time after cold treatment
Time after cold treatment

26. Gene encoding enzymes from octadecanoid pathway & CBF4
transcription factor
Objective II
1st
goal
non-bacterized 26°C
bacterized 26°C
non-bacterized 4°C
bacterized 4°C
200
CBF4
180
160
140
Induction
120
100
80
60
40
20
0
9h
24 h
Time after cold treatment

27. 1. In grapevine plantlets, low temperatures induced the defense-related gene
Objective II
transcripts & cold specific transcription factor CBF4 according to the
phenomenon of priming
1st
goal
Conclusion &
Discussion
2. From analysed genes, except for CBF4, chitinases and glucanases are of
special interest since they exhibit both antifreeze and antifungal activities
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.
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
Barka et al., 2006)

28. Objective II
Characterization of defense mechanisms activated in
fully bacterized plantlets upon exposure to low nonfreezing temperatures
Objective II (2nd goal)
Determination of proline accumulation and analysis of changes in the level
2nd
goal
of lipid peroxidation markers (aldehydes, malondialdehydes) and hydrogen
peroxide

29. Analysis of stress markers (Prolines & Hydrogen peroxide)
Objective II
2nd
goal
b
bacterized 26°C
a
non-bacterized 4°C
a
b
c
a
a
a
b
b
c
c
b
c
c
b
a a
a
a
a a
a a
b b
b b
bacterized 4°C
b
d
d
non-bacterized 26°C
a a
a
c
c
b
b
a
a

30. Analysis of lipid peroxidation markers (Aldehydes & MDA)
Objective II
non-bacterized 26°C
2nd
goal
bacterized 26°C
c
c
non-bacterized 4°C
b
bacterized 4°C
a
a a
a a
a
b
b b
b
c
b
b
c
c
b
a
a
a a
a a
c
b
b
b
a a
c
b
b
a a
a a
a a

31. 1. Apart from gene expression, plant responds to coldness by stress-related
metabolites such as proline, hydrogen peroxide or aldehydes & malondialdehydes, in
Objective II
2nd
goal
similar way to priming phenomenon
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
Conclusion &
acclimation
Discussion
process (Ait Barka et al., 2006). The accumulation of proline in bacterized
plantlets according to the phenomenon of priming, reveals the protective role of
bacteria
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 catalase or peroxidase that
scavenge ROS and help the plant to overcome cold conditions
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 (Barka et al. 2006)

32. Objective II
Characterization of defense mechanisms activated in
fully bacterized plantlets upon exposure to low nonfreezing temperatures
Objective II (3rd goal)
3rd
goal
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

33. Starch deposition & total soluble sugars accumulation
Objective II
d
non-bacterized 26°C
3rd
goal
bacterized 26°C
d
non-bacterized 4°C
d
bacterized 4°C
d
b
a
b
c
a
a
c
c
a
c
b
a b
a
a
b
c
b
b
a
a
b
c
a
a
b
c
c
b c
c
d
b
b
a
a

34. Enzymatic analysis of soluble sugars
Objective II
c
3rd
goal
c
b
b
c
a
a
non-bacterized 26°C
c
c
b
bacterized 26°C
a
b
a
b
a a
a
a
a a
non-bacterized 4°C
bacterized 4°C
b
c
c
d
c
b
c
c
c
b
c
c
c
c
b
b
b
a
a
a
b
a
a
a
a
a
a
a
a
b
b
b
b
a
a
a
a
a
a
a

35. 1. According to our results strain PsJN affects carbohydrate metabolism
Objective I
in grapevine plantlets
in normal growth conditions related with the
stimulation of net photosynthesis (Ait Barka et al., 2006), which may
3rd
goal
contribute to sugar accumulation
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
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

36. CBF4
General
conclusions
B. Phytofirmans is an
ISR-type PGPR able to
prime
PAL
the induction of
StSy
Chit4c
known defense genes
& genes with specific
Chit1b
LOX
role in cold
Gluc
acclimation process
General conclusions
proline
the accumulation of
cryoprotectant
contents
total soluble
sugars
starch
glucose
fructose
sucrose
B. Phytofirmans is an ISRtype PGPR able to potentiate
the physiological response of
cold acclimation and to prime
the grapevine development
and growth upon low nonfreezing temperatures
the faster degradation of
lipid peroxidation and
stress markers
aldehydes
malondialdehyde
hydrogen
peroxide

37. 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
by
Burkholderia
phytofirmans strain PsJNn and for better understanding of
beneficial effects in plants

38. Acknowledgments
Greek Scholarship
Foundation
IKY