Bernard R. Glick - Microorganismos promotores del crecimiento vegetal

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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 …

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.

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  • 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
  • 2. Scientific American Nov. 2011 How can we feed all of the world’s people in the future?
  • 3. 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
  • 4. 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)
  • 5. 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)
  • 6. PGPB promote plant growth directly and indirectlyPGPB promote plant growth directly and indirectly
  • 7. 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
  • 8. 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)
  • 9. 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)
  • 10. 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)
  • 11. •• 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
  • 12. 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)
  • 13. 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)
  • 14. 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
  • 15. 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)
  • 16. 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)
  • 17. 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)
  • 18. 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)
  • 19. 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)
  • 20. 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)
  • 21. Delaying mini-carnation flower senescenceDelaying mini-carnation flower senescence using bacterial endophytesusing bacterial endophytes J. Appl. Microbiol. 113:1139-1144 (2012)
  • 22. Delaying mini-carnation flowerDelaying mini-carnation flower senescence using bacterial endophytessenescence using bacterial endophytes J. Appl. Microbiol. 113:1139-1144 (2012)
  • 23. 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
  • 24. 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
  • 25. 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)
  • 26. 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)
  • 27. 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)
  • 28. 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)
  • 29. 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)
  • 30. 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
  • 31. 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
  • 32. 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)
  • 33. 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
  • 34. 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
  • 35. 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
  • 36. 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)
  • 37. 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
  • 38. 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
  • 39. 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)
  • 40. 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)
  • 41. 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)
  • 42. • 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
  • 43. 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?
  • 44. 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