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Interazioni pianta – azotofissatori Biotecnologie ambientali aa 2012-2013.

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Presentation on theme: "Interazioni pianta – azotofissatori Biotecnologie ambientali aa 2012-2013."— Presentation transcript:

1 Interazioni pianta – azotofissatori Biotecnologie ambientali aa 2012-2013

2 In vista dellesame... Lezioni in ppt disponibili ma suggerisco di utilizzare per lo studio prima testi scritti Programma & letteratura originale Scritto: 2 h (insieme con il prof. Bertoni) 7 o 8 appelli (3 + 3 + 2) Appello subtio dopo Pasqua Segnalate date incompatibili

3 Discutere il ruolo della dormienza del seme nel processo di domesticazione e quale vantaggio / svantaggio conferisce una dormienza estrema o nulla. Fornire un esempio dettagliato di come si riesce ad identificare un gene responsabile della dormienza in una specie di interesse agrario (15 punti) Protocollo di Cartagena: quali sono gli scopi, le richieste e le modalità dellAdvanced Informed Agreement (AIA)? (10 punti) Fitodepurazione: descrivere i vari tipi di fitodepurazione con almeno un esempio di approccio biotecnologico nel dettaglio (5 punti) Esempio di testo desame

4 Le piante coltivate e la sindrome da domesticazione: shattering e dormienza Rischi e benefici ambientali delle piante transgeniche in paragone a quelle convenzionali. Convenzione di Rio, Protocollo di Cartagena e normativa sulle piante create tramite ingegneria genetica Piante per una maggiore sostenibilità ambientale (es. plastiche biodegradabili), per il risanamento (fitodepurazione) e come biosensori di contaminazione. Interazione pianta-microrganismo: le risposte di difesa delle piante e generazione di specie resistenti. Interazione simbiotiche pianta-microrganismo: fissazione dellazoto (batteri azoto fissatori) PROGRAMMA Argomento non trattato

5 Fissazione dellN 2 Haber process now produces 500 million tons of nitrogen fertilizer per year and consumes 3–5% of world natural gas production. It is estimated that half of the protein within a human beings is made of nitrogen that was originally fixed by this process. Only some Bacteria can fix Nitrogen: 100 Mt / yr Fritz Haber (1868 -1934) German chemist, Nobel Prize (1918); developed the process for synthesizing ammonia ( fertilizers and explosives)

6 Fixed nitrogen is one of the limiting factors for plant growth in environments where there is a suitable climate and availability of water to support crops. Nitrogen-fixing cereals would be the breakthrough necessary to underpin sustainable food production for 9 billion people. The production and application of chemical fertiliser is the major source of pollution as well as the major use of energy in agricultural systems. A) Symbiotic diazotrophs - Diazotrophic symbiotic bacteria fix nitrogen only in a specialized structure (nodules) within the host. Examples are Rhizobium, Bradyrhizobium, Frankia… B) Free-living diazotrophs - asymbiotic nitrogen fixers. The asymbiotic nitrogen fixing bacteria can directly convert the gaseous nitrogen to nitrogen rich compounds. On the death of these nitrogen fixers, the soil becomes enriched with nitrogenous compounds thereby serving as biofertilizers e.g. Azobacter sp., Azospirillum sp. http://www.biotechnology4u.com/biotechnology_environment.html Microorganisms which fix nitrogen are called diazotrophs (nitrogen-fixing organisms capable of growth on atmospheric nitrogen as the sole nitrogen source) The ability to fix atmospheric nitrogen via the nitrogenase enzyme complex is restricted to some bacteria. Eukaryotic organisms are only able to obtain fixed nitrogen through their symbiotic interactions with nitrogen-fixing prokaryotes.

7 Asymbiotic nitrogen fixers Groups containing symbiotic fixers Quali batteri fissano lazoto?

8 Example of free living N 2 fixer: Anabaena, cyanobacteria (alghe azzurre) Rhizobium, Bradirhizobium, Azotobacter Symbiotic fixers Rhizobium in free living state Esistono molte variazioni sul tema: ci sono interazioni o simbiosi più o meno strette tra batteri azotofissatori e tanti organismi diversi (piante, funghi, termiti, diatomee...)

9 La fissazione dal punto di vista del batterio: tutti fissano lazoto per mezzo della nitrogenasi http://images.tutorvista.com/content/plant-nutrition/nitrogen-fixation-process.jpeg The nitrogenase complex is comprised of two main functional subunits: dinitrogenase reductase (azoferredoxin) and dinitrogenase (molybdoferredoxin) 4 ATP required per pair of electrons transferred

10 Nitrogenase Fe protein cycle Fe and MoFe proteins of A. vinelandii nitrogenase Dixon & Kahn (2004) Genetic regulation of biological nitrogen fixation. Nature Reviews Microbiology 2, 621-631. La reazione della nitrogenasi è sensibile allossigeno. Come si risolve il problema? Fd red Fd ox

11 The protection is realised by different mechanisms depending on their cellular and physiologic constitutions. - Aerobic bacteria like Azotobacter limit high intracellular oxygen concentrations by high rates of respiratory metabolism in combination with extracellular polysaccharides to reduce oxygen influx. - In some filamentous cyanobacteria, nitrogen fixation is restricted to specialised cells, the heterocysts, which are separated from other cells, and show reduced photosynthetic activity without oxygen production. - Unicellular cyanobacteria combine photosynthesis and nitrogen fixation within the same cell and show a temporary separation of these two pathways where BNF is restricted to the dark period, when the oxygen-levels are low. - In addition to these protections, the concentration of oxygen can be decreased by biochemical pathways like the Mehler-reaction or by special oxygen-scavenging molecules such as cyanoglobin and leghemoglobin, the latter playing a major role in rhizobia-plant interactions Protection of nitrogenase from oxygen

12 spora Le eterocisti si formano quando manca azoto. Hanno parete molto spessa che limita scambi Manca PSII ( non evolvono ossigeno) Anabaena vive in simbiosi con felce Azolla Una strategia per evitare linattivazione delle nitrogenasi: confinare la reazione in una cellula non ossigenica

13 One of the most evolved nitrogen-fixing systems is the root nodule symbiosis (RNS). The symbiosis can be divided into two synchronised but independent programs: bacterial entry and the development of a specialised organ, the root nodule. http://media-3.web.britannica.com/eb-media/38/6538-004-2E138DF9.gif Altra strategia: formazione di noduli (riducono P O 2 )

14 Genetic regulation of biological nitrogen fixation Ray Dixon & Daniel Kahn Nature Reviews Microbiology 2, 621-631 (August 2004) Protection against oxygen is provided by the nodule environment through a cortical diffusion barrier

15 Ulteriore trucco: ridurre P O 2 con una emoglobina Reversible binding of oxygen by leghaemoglobin facilitates oxygen diffusion at low free-oxygen concentrations, supporting bacterioid respiration by a high-affinity terminal oxidase Pink color is due to leghaemoglobin

16 Leghemoglobins accumulate to millimolar concentrations in the cytoplasm of infected plant cells prior to nitrogen fixation and are thought to buffer free oxygen in the nanomolar range, avoiding inactivation of oxygen-labile nitrogenase while maintaining high oxygen flux for respiration. This hypothesis has never been tested in planta. Using RNAi, we abolished symbiotic leghemoglobin synthesis in nodules of the model legume Lotus japonicus.

17 Nodules 14 days after inoculation with rhizobia wild-type LbRNAi Ten-week-old rhizobia- inoculated plants grown in sand without mineral nitrogen Plants grown in grown in soil with nitrogen fertilizer wild-type individuals of two LbRNAi lines Ott T. et al., (2005) Curr Biol. 15:531-5. LbRNAi plants grew normally when fertilized with mineral nitrogen

18 Using a needle-type fiberoptic oxygen microsensor, we found that steady-state levels of free oxygen throughout nodules were higher for the LbRNAi lines than for wild-type controls surface center of nodules Wild-type LbRNAi lines Ott T. et al., (2005) Curr Biol. 15:531-5. Increase in nodule free oxygen, loss of bacterial nitrogenase protein, and absence of SNF

19 rhizosphere Flavonoids nod-gene Inducers (produced by plants) Nod-factor (produced by bacteria) Early events in the Rhizobium-legume symbiosis Rhizobium inducer inhibitors Nodulation is activated by NF application NF recognition at the root surface is likely to be sufficient to activate nodule organogenesis in the root cortex and this must involve a diffusible signal.

20 Nod Factor: a lipo-oligosaccharide nod factors are active on host plants at very low concentration (10 -8 to 10 -11 M) but have no effect on non-host species NFs are chitin (N –acetylglucosamine oligomers) derivatives. The non-reducing end is N -acylated and the reducing end is modified by various molecules.

21 Nod factors inducono allungamento pelo radicale INFEZIONE CONTROLLATA allungamento pelo radicale curvatura pelo radicale

22 root hair beginning to curl Rhizobium cells

23 Nod factors inducono degradazione parete cellulare Si forma il tubetto infettivo per invaginazione della PM Nod factors: sono specie specifici (sia del batterio che della pianta) degradation of cell wall infection thread

24 Cells de-differentiate & divide nodule primordium Ready to receive bacteria from infection thread Controllo ormonale della crescita: Coinvolti auxina, gibberellina ed etilene La formazione del primordio del nodulo avviene lontano dallepidermide

25 Il nodulo matura: forma connessioni vascolari ed esclude O 2 I batteri diventano batterioidi (10 v. più grandi) e iniziano a fissare N 2 Il processo è stato studiato con mutanti incapaci di fissare N 2 perchè bloccati a vari stadi della formazione del nodulo Medicago truncatula-Sinorhizobium meliloti interaction

26 Nodule structure and infection in wild type and Fix mutants Wild type I, meristem central area of the nodules symbiotic cells with intracellular bacteria 50 µm 200 µm 50 µm B: differentiated cells that do not fix nitrogen III: nitrogen fixation C: differentiated plant cells with undifferentiated bacteroids Semi-thin longitudinal sections stained with toluidine blue

27 Plant roots were infected with rhizobia expressing, constitutively, the lacZ gene from the plasmid pXLGD4 and thick nodule sections were stained for β-galactosidase activity. Same mutants as before Per visualizzare meglio linfezione dei batteri si utilizzano batteri con la β-galactosidasi Il processo è controllato da molti geni

28 Metabolismi accoppiati glutaminaasparagina alla pianta legume rhizobia Fixed nitrogen (ammonia) Fixed carbon (malate, sucrose)

29 N2N2 Leg hemo globin O2O2 malato 4O 2 Krebs NADH 1 N 2 8 Ferredox red 8H 2 0 16 ATP nitrogenasi 2 NH 3 + H 2 4 8 Bacterioid metabolism La respirazione aiuta a mantenere bassa la p O 2 Lossidasi terminale ha unaltissima affinità per O 2

30 What are the molecular players involved in nodule formation? Signaling pathways for infection and organogenesis are known NOD factor sensing by Receptor-Like Kinases FLS2 (Flagellin-insensitive 2), a leucine rich repeat (LRR) receptor- like kinase similar to SYMRK

31 Receptor-like kinases Transmembrane proteins with extracellular LRRs and an intracellular kinase domain RLK contain: an extracellular domain, a transmembrane domain and an intracellular protein kinase domain Protein kinase Receptor/sensor

32 Strong similarities between NOD factor and chitin sensing.

33 several non-nodulation mutants are also resistant to colonization by vesicular-arbuscular mycorrhizal fungi (the Myc2 phenotype Plant roots exude strigolactones which induce spore germination and hyphal branching Strigolactone induces seed germination in parasitic plants VAM fungi produce a myc factor Similar signaling pathways are involved in symbiosis with mycorrhizal fungi

34 Myc factors: a mixture of sulphated and non-sulphated simple lipochitooligosaccharides (LCOs) Proposed chemical structures of two major Myc-LCOs General Myc-LCO structure

35 Both symbioses imply an exchange of signalling molecules. Use similar or the same receptors and share several elements of the signal trasduction pathway.

36 SYMRK acts upstream of the Nod factor- and Myc factor-induced calcium spiking

37 Nodulation involves the coordinated development of bacterial infection and nodule organogenesis. Cell division Bacteria are entrapped in a curled root hair infection threads are initiated - Components of the NF signaling pathway are also required for mycorrhizal signaling - Arbuscular mycorrhizal fungi produce NF-like molecules Further division start the nodule primordium A gain-of-function mutation in the cytokinin receptor gene LHK1 of Lotus japonicus was shown to activate spontaneous nodule formation

38 Nodule formation - Modifications of cytokinin levels and application of auxin transport inhibitors causes nodule initiation. - A gain-of-function mutation in the cytokinin receptor gene LHK1 of Lotus japonicus activates spontaneous nodule formation and loss-of- function mutations of LHK1, and its ortholog CRE1 in Medicago truncatula block nodule formation, but allowed bacterial infection. Cytokinin signaling in the root cortex and pericycle is necessary and sufficient for the induction of nodule morphogenesis. It leads to the localized suppression of polar auxin transport, which induces nodule morphogenesis.

39 PLANT GROWTH REGULATORS 1. Endogenous a. Substance produced by a plant that affects the pattern of growth and development. b. Production by the plant is regulated by the environment. 2. Exogenous a. Substance applied to the plant that alters growth and development in the same way that endogenous substances do. b. May be the same or different chemically from the endogenous substance Hormone a. Substance that acts in very low concentration (micro-molar or less) b. Produced in one part of plant and act in another (translocatable) c. Has the same response in many different plant species

40 Primary 1. Auxins 1. Auxins 2. Cytokinins 2. Cytokinins 3. Gibberellins 3. Gibberellins 4. Abscisic Acid 4. Abscisic Acid 5. Ethylene 5. Ethylene Secondary - newly discovered 1. Jasmonic Acid 1. Jasmonic Acid 2. Brassinosteroids 2. Brassinosteroids 3. Juglone 3. Juglone 4. Salicylic Acid 4. Salicylic Acid 5. Polyamines 5. Polyamines Others - not yet confirmed or understood 1. Peptide Hormones (animals maybe plants) 1. Peptide Hormones (animals maybe plants) 2. Oligosaccharides (cell wall signaling) 2. Oligosaccharides (cell wall signaling) 3. Phospholipids (inositol phosphates, diacylglycerides) 3. Phospholipids (inositol phosphates, diacylglycerides) 4. mRNA or Protein 4. mRNA or Protein Florigen (floral induction) Florigen (floral induction)

41 A Survey of Plant Hormones

42 Lormone AUXINA Triptofano

43 La scoperta dellAuxina Lesperimento dimostra che il sito di percezione è diverso dal sito che risponde (dove avviane la curvatura). ci deve essere la trasmissione di un segnale dallapice alla base

44 Materiale sperimentale: coleottile

45 Auxin Discovery

46 Il segnale è un fattore diffusibile che si muove verso la base del coleottile Test biologico quantitativo (permette una misura della quantità dellauxina)

47 Growth and morphogenesis of root in A. thaliana. Ben Scheres group: Molecular Genetics Group, Department of Biology, Utrecht University

48 http://www.uu.nl/SiteCollectionImages/Fac_Beta/Molecular%20Genetics/DR5-GUS1.jpg An auxin maximum in the primary root and at the emerging lateral roots costrutto reporter DR5::GUS http://ars.els-cdn.com/content/image/1-s2.0-S0092867403009243-gr1.jpg

49 costrutto reporter DR5::GFP costrutto reporter pPIN1::PIN1::GFP costrutto reporter pPIN2::PIN2::GFP costrutto reporter pPIN7::PIN7::GFP http://www.nature.com/nature/journal/v457/n7233/images/nature07597-f3.2.jpg http://www.idw-online.de/pages/de/newsimage?id=218&size=screen pin1 mutant (arabidopsis)

50 Arabidopsis root model The model consists of several cell files, each composed of different cells with different parameters (transpor rate, concentration...) which are set according to experimental data Most remarkably: the model describes root behavior quite well! e.g. distribution of different auxin transporters (PIN proteins)

51 During the initiation and maintenance of the root apical meristem, cytokinin and auxin function in an antagonistic manner and appear to act in different zones of the meristem Auxin accumulates at the tip of the root apical meristem, and this is crucial to maintain cell division cytokinin acts at the boundary between the proximal meristem and the elongation zone, and is associated with the transition from cell division to cell

52 Auxin and cytokinin during root meristem development Lateral root initiation and nodule formation share several characteristics Cytokinin signaling during nodulation is restricted to the pericycle and cortical cells where cell divisions occur during nodule initiation Localized accumulations of auxin in the pericycle mark the site where the lateral root emerges. Cytokinin can suppress lateral root emergence by blocking the localized accumulations of auxin, probably through the suppression of PINs. During nodule development, localized cytokinin signaling in the root cortex is necessary and sufficient for the initiation of the nodule primordia Localized suppression of polar auxin transport creates a minimum of auxin which induce nodule morphogenesis

53 In conclusion We know a lot about symbiotic interactions with nitrogen fixing bacteria - Requires an exchange of signalling molecules between plant and bacterium. - Use similar or the same receptors for the bacterial signal. - Share several elements of the signal trasduction pathway. - Induce the formation of nodule primordium in a manner similar to secondary root formation As with many other situations, all the knowledge gained by fundamental research should allow the creation of plants requiring less input for a more environmentally sustainable agriculture.

54 Bibliografia Dixon & Kahn (2004) Genetic regulation of biological nitrogen fixation Nature Reviews Microbiology 2:621-631. Oldroyd et al., (2011) The Rules of Engagement in the Legume-Rhizobial Symbiosis. Annu. Rev. Genet. 45:119-44. Ott T. et al., (2005) Symbiotic leghemoglobins are crucial for nitrogen fixation in legume root nodules but not for general plant growth and development. Curr Biol. 15:531-5. Maunoury (2010) Differentiation of Symbiotic Cells and Endosymbionts in Medicago truncatula Nodulation Are Coupled to Two Transcriptome-Switches. PLoS One. 5:e9519. Kneip (2007) Nitrogen fixation in eukaryotes – New models for symbiosis BMC Evolutionary Biology 2007, 7:55. Parniske M. (2008) Arbuscular mycorrhiza: the mother of plant root endosymbioses. Nat Rev Microbiol. 6:763-75. Maillet F. et al., (2011) Fungal lipochitooligosaccharide symbiotic signals in arbuscular mycorrhiza. Nature. 469:58-63. Endre G. et al. (2002) A receptor kinase gene regulating symbiotic nodule development. Nature. 417:962-6.

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56 Infection strategies Root hair infection (RHI)lateral root base (LRB)nodal root base (NRB)


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