Los días 20 y 21 de mayo de 2014, la Fundación Ramón Areces organizó el Simposio Internacional 'Microorganismos beneficiosos para la agricultura y la protección de la biosfera' dentro de su programa de Ciencias de la Vida y de la Materia.

1. Plant Growth-PromotingPlant Growth-Promoting
MicroorganismsMicroorganisms
Bernard R. GlickBernard R. Glick
Department of BiologyDepartment of Biology
University of WaterlooUniversity of Waterloo
Waterloo, Ontario, CanadaWaterloo, Ontario, Canada
glick@sciborg.uwaterloo.caglick@sciborg.uwaterloo.ca

3. Scientific American Nov. 2011
How can we feed all of the
world’s people in the future?

4. Crop productivity may be increasedCrop productivity may be increased
through the use of either i) transgenicthrough the use of either i) transgenic
plants or ii) the application of plantplants or ii) the application of plant
growth-promoting bacteriagrowth-promoting bacteria

5. Plant Growth-Promoting BacteriaPlant Growth-Promoting Bacteria
Soil bacteria that facilitate plant growthSoil bacteria that facilitate plant growth
1.1. In association with roots (rhizospheric)In association with roots (rhizospheric)
2.2. On leaves or flowers (phyllospheric)On leaves or flowers (phyllospheric)
3.3. Within plant tissues (endophytic and symbiotic)Within plant tissues (endophytic and symbiotic)
Can J. Microbiol. 41: 109-117 (1995)Can J. Microbiol. 41: 109-117 (1995)

6. Plant growth as aPlant growth as a
function of agefunction of age
Arrows indicate stressArrows indicate stress
onset which causes growthonset which causes growth
to slow or stopto slow or stop
Actual yield variesActual yield varies
according to the numberaccording to the number
and intensity of the stressesand intensity of the stresses
that a plant experiencesthat a plant experiences
Eur. J. Plant Pathol. 119: 329-339 (2007)Eur. J. Plant Pathol. 119: 329-339 (2007)

7. PGPB promote plant growth directly and indirectlyPGPB promote plant growth directly and indirectly

8. Cleavage of ACC toCleavage of ACC to αα-ketobutyrate-ketobutyrate
and ammonia by ACC deaminaseand ammonia by ACC deaminase
Biochim. Biophys. Acta 1703: 11-19 (2004)Biochim. Biophys. Acta 1703: 11-19 (2004)
4

9. ACC Deaminase PropertiesACC Deaminase Properties
•• Common in soil bacteria and fungiCommon in soil bacteria and fungi
•• Requires pyridoxal phosphateRequires pyridoxal phosphate
•• Native form is a dimer or trimerNative form is a dimer or trimer
•• Monomer is 35-45 kDMonomer is 35-45 kD
•• Km for ACC is ~1-15 mMKm for ACC is ~1-15 mM
•• Induced by low levels of ACCInduced by low levels of ACC
•• Sulfhydryl enzymeSulfhydryl enzyme
•• Cytoplasmically localizedCytoplasmically localized
FEMS Microbiol. Lett. 251: 1-7 (2005)FEMS Microbiol. Lett. 251: 1-7 (2005)

10. Interaction of aInteraction of a
bacterium containingbacterium containing
ACC deaminase withACC deaminase with
a plant decreasesa plant decreases
both formation ofboth formation of
stress ethylene andstress ethylene and
environmentalenvironmental
damage to the plantdamage to the plant
J. Theor. Biol. 190: 63-68 (1998)J. Theor. Biol. 190: 63-68 (1998)

11. Stress ethyleneStress ethylene
before and afterbefore and after
treatment with antreatment with an
ACC deaminase-ACC deaminase-
containingcontaining
bacteriumbacterium
Eur. J. Plant Pathol. 119: 329-339 (2007)Eur. J. Plant Pathol. 119: 329-339 (2007)

12. •• Promotes root initiationPromotes root initiation
•• Inhibits root elongationInhibits root elongation
•• Promotes fruit ripeningPromotes fruit ripening
•• Promotes flower wiltingPromotes flower wilting
•• Stimulates seed germinationStimulates seed germination
•• Promotes leaf abscissionPromotes leaf abscission
•• Response to biotic and abiotic stressResponse to biotic and abiotic stress
•• Activates hormone synthesisActivates hormone synthesis
•• InhibitsInhibits RhizobiaRhizobia nodule formationnodule formation
•• Inhibits mycorrhizae interactionInhibits mycorrhizae interaction
How does ethylene affect plants?How does ethylene affect plants?
Abeles et al., 1992, Ethylene in Plant Biology

13. Effect of bacteria containing ACCEffect of bacteria containing ACC
deaminase on canola root lengthdeaminase on canola root length
Can. J. Microbiol.Can. J. Microbiol.
44: 833-843 (1998)44: 833-843 (1998)

14. Plant protection from stress ethylenePlant protection from stress ethylene
by ACC deaminase-containing PGPBby ACC deaminase-containing PGPB
•• PhytopathogensPhytopathogens
•• High saltHigh salt
•• FloodingFlooding
•• DroughtDrought
•• Heavy metalsHeavy metals
•• Temperature extremesTemperature extremes
•• Organic contaminantsOrganic contaminants
•• NematodesNematodes
Adv. Appl. Microbiol. 56: 291-312 (2004)Adv. Appl. Microbiol. 56: 291-312 (2004)

15. Mung bean ± PGPBMung bean ± PGPB Castor bean +Castor bean +
Agro ± PGPBAgro ± PGPB
Canola + salt ± PGPBCanola + salt ± PGPB
Tomato + flooding ± PGPBTomato + flooding ± PGPB
0
10
20
30
40
50
60
Control GR12-2 ARV8
Dryweight,mgDryweight,mg
Tomato + drought ± PGPBTomato + drought ± PGPB

16. Treatment Weight of Rotted Potatoes, g
Erwiniacarotovora 15.6 ± 3.4
Erwiniacarotovora+ Biocontrol
bacterium
14.5 ± 2.8
Erwiniacarotovora + Biocontrol
bacterium + ACC deaminase
7.5 ± 2.3
PGPB with ACC deaminase increase the biomass ofPGPB with ACC deaminase increase the biomass of
pythium-treated cucumber plants and decrease thepythium-treated cucumber plants and decrease the
damage to potatoes bydamage to potatoes by Erwinia carotovoraErwinia carotovora
Can. J. Microbiol. 46: 898-907 (2000)

17. Pine seedlings (A) with no additions, (B) + pinewood nematode (PWN),
C) + PWN + P. putida UW4/AcdS, (D) + PWN + wild-type P. putida UW4
Protection of Pine Seedlings from Pinewood DiseaseProtection of Pine Seedlings from Pinewood Disease
Caused by Pinewood NematodeCaused by Pinewood Nematode
Biocontrol 58:427-433 (2013)

18. Achromobacter piechaudiiAchromobacter piechaudii ARV8ARV8
increases tomato plant salt toleranceincreases tomato plant salt tolerance
Plant Physiol. Biochem. 42: 565-572 (2004)Plant Physiol. Biochem. 42: 565-572 (2004)

19. Isolate
Bacteria
ACC deaminase
IAA synthesis
Siderophores
Phosphate solubilization
Salt tolerance
Plant growth promotion
Choose “best” strains
Construct AcdS mutants
Soil samples
Test wild-type and mutant
bacteria for growth promotion
in soil containing salt
Selection scheme for new endophytic PGPB
Appl. Soil. Ecol. 61:217-224 (2012)

20. Growth of 11-week-old tomato plants +Growth of 11-week-old tomato plants +
185 mM salt + bacterial endophytes185 mM salt + bacterial endophytes
Plant Physiol. Biochem. 80:160-167 (2014)

21. Growth of 11-week-old tomato plants + salt +Growth of 11-week-old tomato plants + salt +
bacterial endophytesbacterial endophytes
A = no bacteria; B = endophyte 1 wild-type; C = endophyte 1 mutant;
D = endophyte 2 wild-type; E = endophyte 2 mutant
Plant Physiol. Biochem. 80:160-167 (2014)

22. Delaying mini-carnation flower senescenceDelaying mini-carnation flower senescence
using bacterial endophytesusing bacterial endophytes
J. Appl. Microbiol. 113:1139-1144 (2012)

23. Delaying mini-carnation flowerDelaying mini-carnation flower
senescence using bacterial endophytessenescence using bacterial endophytes
J. Appl. Microbiol. 113:1139-1144 (2012)

24. Rhizospheric
PGPB specific
genes
Endophytic
PGPB specific
genes
Genes common to
Rhizospheric and
Endophytic PGPB
Genes involved in endophytic behavior
J. Theor. Biol. 343:193-198 (2014)
Nearly all of the genes identified by this bioinformatics
procedure encode functions previously suggested
in other studies to be involved in endophytic behavior

25. Transgenic,Transgenic, rolDrolD ACC deaminase, plantsACC deaminase, plants
(tomato and canola) behave similarly to(tomato and canola) behave similarly to
PGPB-treated plants in response toPGPB-treated plants in response to
various environmental stressesvarious environmental stresses

26. Some, but not all, strains ofSome, but not all, strains of RhizobiumRhizobium spp.spp.
contain ACC deaminasecontain ACC deaminase
•• R. leguminosarumR. leguminosarum bv.bv. viciaeviciae yesyes
•• R. leguminosarumR. leguminosarum bv.bv. phaseoliphaseoli yesyes
•• R. leguminosarumR. leguminosarum bv.bv. trifoliitrifolii nono
•• R. spR. sp. Designati. Designati nono
•• R. hedysariR. hedysari yesyes
•• M. ciceriM. ciceri nono
•• R. radicicolaR. radicicola nono
•• M. lotiM. loti no/yesno/yes
•• S. melilotiS. meliloti nono
Anton. von Leeuwenhoek 83: 285-291 (2003)Anton. von Leeuwenhoek 83: 285-291 (2003)

27. Pea plants inoculated withPea plants inoculated with
differentdifferent R.R. leguminosarumleguminosarum strainsstrains
Wild-Wild-
TypeType
acdRacdR KOKO acdSacdS KOKO acdSacdS ++++++
Appl. Environ. Microbiol. 69: 4396-4402 (2003)Appl. Environ. Microbiol. 69: 4396-4402 (2003)

28. Acquisition of ACC deaminase byAcquisition of ACC deaminase by S. melilotiS. meliloti
increases nodulation and biomass of alfalfaincreases nodulation and biomass of alfalfa
Appl. Environ. Microbiol. 70: 5891-5897 (2004)Appl. Environ. Microbiol. 70: 5891-5897 (2004)

29. Mesorhizobium ciceri LMS-1 expressing an exogenous
ACC deaminase increases its nodulation abilities and
chickpea plant resistance to soil constraints
Wild-type Transformant
Lett. Appl. Microbiol. 55:15-21 (2012)

30. PhytoremediationPhytoremediation
The use of plants (and bacteria) to sequester,The use of plants (and bacteria) to sequester,
stabilize or break down environmentalstabilize or break down environmental
contaminants (usually either organics such ascontaminants (usually either organics such as
PCBs and PAHs, or metals)PCBs and PAHs, or metals)

31. Phytoremediation problem:
Environmental contaminants inhibit plant
growth even with plants that are
hyperaccumulators
One solution: Use PGPB to reduce stress
and facilitate plant growth during
phytoremediation
Another solution: Use transgenic plants
that more efficiently sequester metals or
organic contaminants and reduce stress

32. Bacterial siderophores help plants acquire ironBacterial siderophores help plants acquire iron
in the presence of metal contaminationin the presence of metal contamination
Six-coordinate iron-
siderophore complex

33. ACC deaminase-containing PGPB promotes growthACC deaminase-containing PGPB promotes growth
ofof Brassica junceaBrassica juncea in nickel contaminated soilin nickel contaminated soil
Can. J. Microbiol. 46: 237-245 (2000)Can. J. Microbiol. 46: 237-245 (2000)

34. PGPB with ACC deaminase promotes growthPGPB with ACC deaminase promotes growth
of tobacco in copper-contaminated soilof tobacco in copper-contaminated soil
Unpublished dataUnpublished data

35. PGPB with ACC deaminase promote thePGPB with ACC deaminase promote the
growth of canola in PAH-contaminated soilgrowth of canola in PAH-contaminated soil
Can J. Microbiol. 51: 1061-1069 (2005)Can J. Microbiol. 51: 1061-1069 (2005)
PAHsPAHs

36. ACC deaminase-containing PGPB are asACC deaminase-containing PGPB are as
effective in a contaminated field as in the labeffective in a contaminated field as in the lab
Barley/rye mixtureBarley/rye mixture with or without PGPBwith or without PGPB
WithoutWithout WithWith WithWith WithoutWithout
Unpublished dataUnpublished data

37. Phytoremediation of petroleum-Phytoremediation of petroleum-
contaminated soil ± ACC deaminase-contaminated soil ± ACC deaminase-
containing PGPBcontaining PGPB
Microchem. J. 81: 139-147 (2005)Microchem. J. 81: 139-147 (2005)

38. PGPB relieve growth inhibition of rice fromPGPB relieve growth inhibition of rice from
residual herbicides from the previous seasonresidual herbicides from the previous season
+ PGPB+ PGPB
–– PGPBPGPB
Unpublished dataUnpublished data

39. How does the PGPBHow does the PGPB
P. putidaP. putida UW4 affect mRNAUW4 affect mRNA
in canola shoots and roots?in canola shoots and roots?
What is the role of bacterialWhat is the role of bacterial
ACC deaminase in canolaACC deaminase in canola
mRNA expression?mRNA expression?
and

40. B C
How do ACC deaminase-containing PGPBHow do ACC deaminase-containing PGPB
change canola gene expression?change canola gene expression?
BB auxin response factors increase upon adding wild-typeauxin response factors increase upon adding wild-type
CC stress response genes increase upon adding mutantstress response genes increase upon adding mutant
Mol. Plant-Microbe Interact. 25:668-676 (2012)Mol. Plant-Microbe Interact. 25:668-676 (2012)

41. Pseudomonas putida UW4 Genome
The outer strand is the + strand
tRNA is in green and rRNA is in red
Black indicates deviation from average GC content
Inner circle is GC skew; leading strand has G>C
• GC content: 60.05%
• rRNA genes: 22
• tRNA genes: 72
• Protein coding genes:
5570
• % coding bases: 87.7%
• ACC deaminase - PGPBACC deaminase - PGPB
• Siderophores - PGPBSiderophores - PGPB
• IAA biosynthesis - PGPBIAA biosynthesis - PGPB
• Antifreeze protein - ColdAntifreeze protein - Cold
• Cold shock - ColdCold shock - Cold
• Trehalose – Salt & DroughtTrehalose – Salt & Drought
• Metal toleranceMetal tolerance
PLoS ONE 8(3): e58640 (2013)

42. IAA biosynthesis in Pseudomonas sp. UW4
D-ribulose-5P
D-ribose-5P
PRPP
P
Pu
Py
His
ACC + H2O ! -ketobutyrate + NH3
+
IAN
IAM IAA
Trp
IAA synthesis
Glycogen
Maltodextrin
Maltooligosyl-trehalose Diac
Acetoin
ADP
ADP
ATP
de/
e
ADP
ATP-
L-
e/
e
ADP
ATP
Fe3+
um
H4+
de
IAOx
cysteineSerine O-acetylserine
1. Two potential IAA biosynthesis pathways, the IAM and IAN pathways,
were identified in the genome of UW4
2. Biochemical characterization of some of these enzymes has confirmed
that these putative pathways are operative in UW4 (in press)
3. Several other sequenced Pseudomonas genomes appear to have
similar IAA synthesis pathways compared to UW4
PLoS ONE 8(3): e58640 (2013)

43. • Ethylene feedbackEthylene feedback
loop prevents too muchloop prevents too much
ethylene from beingethylene from being
synthesized by IAA-synthesized by IAA-
producing bacteriaproducing bacteria
• ACC deaminaseACC deaminase
lowers ethylenelowers ethylene
inhibition of plantinhibition of plant
growthgrowth andand increasesincreases
IAA flux, both of whichIAA flux, both of which
promote plant growthpromote plant growth
In press
RevisedRevised model of ACC deaminase andmodel of ACC deaminase and
IAA promoting plant growthIAA promoting plant growth

44. As a consequence of the fundamental knowledgeAs a consequence of the fundamental knowledge
of PGPB modes of action that has been elaboratedof PGPB modes of action that has been elaborated
over the past 10-20 years, this technology isover the past 10-20 years, this technology is
currently accessible for use in agriculture,currently accessible for use in agriculture,
horticulture, and environmental cleanuphorticulture, and environmental cleanup
technologies in both the developed and thetechnologies in both the developed and the
developing world.developing world.
Future prospects?Future prospects?

45. Contributors to the work discussedContributors to the work discussed
Shimon MayakShimon Mayak Donna PenroseDonna Penrose
Saleh ShahSaleh Shah Barbara GrichkoBarbara Grichko
Henry BurdHenry Burd Saleema SalehSaleema Saleh
Wenbo MaWenbo Ma Trevor CharlesTrevor Charles
Frederique GuinelFrederique Guinel Jennifer StearnsJennifer Stearns
Peter PaulsPeter Pauls Barbara MoffattBarbara Moffatt
XiaoDong HuangXiaoDong Huang George DixonGeorge Dixon
Bruce GreenbergBruce Greenberg Tsipi TiroshTsipi Tirosh
Yola GurskaYola Gurska Brendan McConkeyBrendan McConkey
Gina HolguinGina Holguin Chunxia WangChunxia Wang
Nikos HontzeasNikos Hontzeas Lucy ReedLucy Reed
Zhenyu ChengZhenyu Cheng Yoav BashanYoav Bashan
Jin DuanJin Duan John HeikkilaJohn Heikkila
Youai HaoYouai Hao Elisa GamaleroElisa Gamalero
Graziella BertaGraziella Berta Jiping LiJiping Li
Stephanie SebestianovaStephanie Sebestianova Cheryl PattenCheryl Patten
Leonid CherninLeonid Chernin Brendan McConkeyBrendan McConkey
Solange OliveiraSolange Oliveira Francisco NasimentoFrancisco Nasimento
Daiana DucaDaiana Duca Clarisse BrigidoClarisse Brigido
Shimaila AliShimaila Ali Guido LinguaGuido Lingua