Interazioni pianta – azotofissatori

1. Biotecnologie ambientaliaa 2013-2014 Interazioni pianta – azotofissatori

2. In vista dell’esame... 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: Jan-Feb, 3: Jun-Jul + 2: Sept e subito dopo Pasqua Segnalate date incompatibili

3. Esempio di testo d’esame 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 dell’Advanced Informed Agreement (AIA)? (10 punti) Descrivere la strategia di resistenza mediata da PAMP con almeno un esempio di approccio biotecnologico per migliorare la resistenza ai patogeni (5 punti)

4. PROGRAMMA • 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 dell’azoto (batteri azoto fissatori) Argomento non trattato

5. Fissazione dell’N2 Haber process now produces 100 million tons of ammonia (500 Mt 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. The production and application of chemical fertiliser is the major source of pollution as well as the major use of energy in agricultural systems. Nitrogen-fixing cereals would be the breakthrough necessary to underpin sustainable food production for 9 billion people. 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. Microorganisms which fix nitrogen are called diazotrophs (nitrogen-fixing organisms capable of growth on atmospheric nitrogen as the sole nitrogen source) 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

7. Asymbiotic nitrogen fixers Quali batteri fissano l’azoto? Groups containing symbiotic fixers

8. Example of free living N2 fixer: Anabaena, cyanobacteria (alghe azzurre) 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...) Rhizobium, Bradirhizobium, Azotobacter Symbiotic fixers Rhizobium in free living state

9. La fissazione dal punto di vista del batterio: tutti fissano l’azoto per mezzo della nitrogenasi 4 ATP richiesti per coppia di elettroni transferiti 8 e- richiesti in totale per la riduzione di 1 molecola di N2 6 e- vanno sull’azoto, 2 vengono rilasciati come H2 The nitrogenase complex is comprised of two main functional subunits: dinitrogenase reductase (azoferredoxin, Fe-protein) and dinitrogenase (molybdoferredoxin, MoFe-protein) http://images.tutorvista.com/content/plant-nutrition/nitrogen-fixation-process.jpeg

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

11. Protection of nitrogenase from oxygen 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

12. Una strategia per evitare l’inattivazione delle nitrogenasi: confinare la reazione in una cellula non ossigenica 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

13. Altra strategia: formazione di noduli (riducono PO2) 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 (after mutual recognition) - development of a specialised organ, the root nodule. http://media-3.web.britannica.com/eb-media/38/6538-004-2E138DF9.gif Come la formazione del nodulo facilita la reazione della nitrogenasi?

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 PO2 con una emoglobina Pink color is due to leghaemoglobin Reversible binding of oxygen by leghaemoglobin facilitates oxygen diffusion at low free-oxygen concentrations, supporting bacterioid respiration by a high-affinity terminal oxidase

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

18. center of nodules surface 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 LbRNAi lines Wild-type Increase in nodule free oxygen, loss of bacterial nitrogenase protein, and absence of SNF Ott T. et al., (2005) Curr Biol. 15:531-5.

19. inducer inhibitors Flavonoids nod-gene Inducers (produced by plants) Nod-factor (produced by bacteria) Early events in the Rhizobium-legume symbiosis 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. rhizosphere Rhizobium

20. Chitin: N –acetylglucosamine Nod Factor: a lipo-oligosaccharide NFs are chitin (N –acetylglucosamine oligomers) derivatives. The non-reducing end is N -acylated and the reducing end is modified by various molecules. 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

21. INFEZIONE CONTROLLATA Nod factors inducono allungamento pelo radicale 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 degradation of cell wall infection thread Nod factors: sono specie specifici (sia del batterio che della pianta)

24. Rhizobium encoding GFP from jellyfish as a marker Infection thread (From Quaedvlieg et al. Plant Mol. Biol. 37: 715-727, 1998)

25. 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 dall’epidermide

26. Il nodulo matura: forma connessioni vascolari ed esclude O2 I batteri diventano batterioidi (10 v. più grandi) e iniziano a fissare N2 Il processo è stato studiato con mutanti incapaci di fissare N2 perchè bloccati a vari stadi della formazione del nodulo  Medicago truncatula-Sinorhizobium meliloti interaction

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

28. Per visualizzare meglio l’infezione dei batteri si utilizzano batteri con la β-galactosidasi Il processo è controllato da molti geni 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

29. Two waves of transcriptional reprogramming involving the repression and the massive induction of hundreds of genes were observed during wild-type nodule formation. • The dominant features of this “nodule-specific transcriptome” were • the repression of plant defense-related genes, • the transient activation of cell cycle and protein synthesis genes at the early stage of nodule development and • the activation of the secretory pathway along with a large number of transmembrane and secretory proteins or peptides throughout organogenesis

30. Fixed nitrogen (ammonia) Fixed carbon (malate, sucrose) glutamina asparagina alla pianta Metabolismi accoppiati legume rhizobia

31. Bacterioid metabolism malato Leg hemo globin Krebs La respirazione aiuta a mantenere bassa la pO2 NADH 8 4 4O2 O2 8 Ferredoxred L’ossidasi terminale ha un’altissima affinità per O2 16 ATP 8H20 nitrogenasi N2 1 N2 2 NH3 + H2

32. What are the molecular players involved in nodule formation? NOD factor sensing by Receptor-Like Kinases Nodulation is activated by NF application and by gain-of-function mutations of CCaMK. 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. Signaling pathways for infection and organogenesis are known

33. Receptor-like kinases Transmembrane proteins with extracellular LRRs and an intracellular kinase domain Receptor/sensor Protein kinase FLS2 (Flagellin-insensitive 2), is a leucine rich repeat (LRR) receptor-like kinase similar to SYMRK RLK contain: 1) an extracellular domain, 2) a transmembrane domain and 3) an intracellular protein kinase domain

34. Strong similarities between NOD factor and chitin sensing.

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

36. Sezione di radice al microscopio con contrasto di fase Populus tremuloides ectomicorrize. La rete di ife avvolge le cellule epidermiche. Le ramificazioni aumentano la superficie di contatto tra il fungo e la radice http://www.ffp.csiro.au/research/mycorrhiza/ecm.html

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

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

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

40. 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 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.

41. Punti in comune tra le due simbiosi: - Segnali emessi dalla pianta attivano/attirano il simbiote microbico - In risposta i simbionti (batterio o fungo) emettono segnali costituiti da lipo-oligosaccaridi. L’unità base dello zucchero è la chitina (N-actil-glucosamina). Questi segnali sono percepiti a livello della superficie e dentro la radice attivando un programma di sviluppo che richiede divisioni cellulari secondo direzioni definite Come si sviluppa normalmente la radice? Come la simbiosi modifica il suo sviluppo?

42. 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

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

44. A Survey of Plant Hormones

45. L’ormone AUXINA Triptofano

46. La scoperta dell’Auxina L’esperimento dimostra che il sito di percezione è diverso dal sito che risponde (dove avviane la curvatura).  ci deve essere la trasmissione di un segnale dall’apice alla base

47. Materiale sperimentale: coleottile

48. Auxin Discovery

49. Il segnale è un fattore diffusibile che si muove verso la base del coleottile Auxin Discovery Test biologico quantitativo (permette una misura della quantità dell’auxina)

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