Physiological responses of Physiological responses of Burkholderia Burkholderia phytofirmans phytofirmans strain strain PsJN PsJN colonized plantlets of grapevine ( colonized plantlets of grapevine ( Vitis Vitis vinifera vinifera L.) to L.) to low non low 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
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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
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