Unité de Recherche Vignes et Vins de
Champagne Stress et Environnement
UFR Sciences Exactes et Naturelles

Laboratoire
de ...
Sustainable development
Literature
Review

The idea of a world where people protect the environment
as they carry out thei...
Plant growth promoting rhizobacteria (PGPR) & plants
Literature
Review

Plant growth promotion
• Nitrogen fixation
• Solub...
ISR & signaling pathways
Literature
Review

A state of increased defensive capacity developed in
ISR-

by inducing PGPR
th...
Systemic resistance by primed physiological responses
Literature
Review

Systemic response induced by

Pathogens

coloniza...
Priming mechanism : Causes and Effects
Literature
Review

a) Primed physiological stage

Cold acclimation

Pathogens,
PGPR...
Burkholderia phytofirmans strain PsJN
Literature
Review

A member of the genus Burkholderia belongs to the ß-Proteobacteri...
Grapevine – B. phytofirmans strain PsJN interaction
Literature
Review

Significant capacity of bacterized plantlets to
wit...
Plant sensitivity in low non-freezing temperatures
(0°C - 15°C )
Literature
review

Plants
Tropical &
subtropical origin

...
Chilling injury in sensitive plants
Literature
review

Cell membranes
phase
LiquidSolid gel
Crystalline
transition

Return...
AOS accumulation induces chilling injuries in sensitive plants
Literature
Review
Plant biology
in cold
Sensitive
plants

P...
Responses of insensitive plants to low non-freezing temperatures
Literature
Review

Accumulation of sugars
and prolines

M...
The process of cold acclimation & the signal
transduction pathways
Literature
Review

Induction of
antioxidant
systems

Co...
Accumulation of cryoprotectant contents in acclimated plants
Literature
Review
Plant biology
in cold
Insensitive
plants

C...
Despite the available information by previous studies,
several questions remained regarding the beneficial
interaction bet...
The three objectives of the project

Objective I

Objective II

Study of response of grapevine plantlets after root
inocul...
Objective I

Study of response of grapevine plantlets after root
inoculation by Bulkholderia phytofirmans strain
PsJN
Obje...
Vitis vinifera L. cv. Chardonnay clone 7573

16-h fluorescent light at 26°C

Objective I

micro-cuttings
1 nodal explant

...
Genes encoding enzymes from phenylopropanoid
& octadecanoid pathways
Objective I

Stilbene synthase (VvStSy)
Induction

40...
Genes encoding for pathogenesis-related proteins (PRs)
Objective I

Induction

Chitinase 4c (VvChit4c)
10
8
6
4
2
0
0

12
...
i)

Induction of defense mechanism after root inoculation

Objective I

PRs

ii) Induction of systemic responses by strain...
Objective II

Characterization of defense mechanisms activated in
fully bacterized plantlets upon exposure to low nonfreez...
Vitis vinifera L. cv. Chardonnay clone 7573
16-h light/
8-h dark at
26°C

microcuttings
1 node

Objective II

propagation
...
Genes coding enzymes from phenylopropanoid pathways

Objective II

1st
goal

non-bacterized 26°C
bacterized 26°C
non-bacte...
Genes encoding for pathogenesis-related proteins (PRs)
Objective II

1st
goal

non-bacterized 26°C
bacterized 26°C
non-bac...
Gene encoding enzymes from octadecanoid pathway & CBF4
transcription factor
Objective II

1st
goal
non-bacterized 26°C
bac...
1. In grapevine plantlets, low temperatures induced the defense-related gene
Objective II

transcripts & cold specific tra...
Objective II

Characterization of defense mechanisms activated in
fully bacterized plantlets upon exposure to low nonfreez...
Analysis of stress markers (Prolines & Hydrogen peroxide)
Objective II

2nd
goal

b

bacterized 26°C
a

non-bacterized 4°C...
Analysis of lipid peroxidation markers (Aldehydes & MDA)
Objective II
non-bacterized 26°C

2nd
goal

bacterized 26°C
c

c
...
1. Apart from gene expression, plant responds to coldness by stress-related
metabolites such as proline, hydrogen peroxide...
Objective II

Characterization of defense mechanisms activated in
fully bacterized plantlets upon exposure to low nonfreez...
Starch deposition & total soluble sugars accumulation
Objective II
d

non-bacterized 26°C

3rd
goal

bacterized 26°C
d

no...
Enzymatic analysis of soluble sugars

Objective II
c

3rd
goal

c
b

b

c

a

a

non-bacterized 26°C

c

c

b

bacterized ...
1. According to our results strain PsJN affects carbohydrate metabolism
Objective I

in grapevine plantlets

in normal gro...
CBF4
General
conclusions

B. Phytofirmans is an
ISR-type PGPR able to
prime

PAL
the induction of
StSy
Chit4c
known defens...
Future prospects

Prospect I
Investigation of primed- physiological state of V. vinifera L. induced
by Burkholderia phytof...
Acknowledgments

Greek Scholarship
Foundation
IKY
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Physiological responses of_burkholderia_phytofirmans_strain_ps_jn_colonized_plantlets_of_grapevine__vitis_vinifera_l.__to_low_non-freezin

  1. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 38. Acknowledgments Greek Scholarship Foundation IKY

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