Interazioni pianta – azotofissatori

Biotecnologie ambientaliaa 2012-2013

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 + 3 + 2)• Appello subtio dopo Pasqua• Segnalate date incompatibili

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)

Fitodepurazione: descrivere i vari tipi di fitodepurazione con almeno un esempio di approccio biotecnologico nel dettaglio (5 punti)

Esempio di testo d’esame

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)

PROGRAMMA

Argomento non trattato

Fissazione dell’N2

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)

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.

Asymbiotic nitrogen fixersGroups containing symbiotic fixers

Quali batteri fissano l’azoto?

Example of free living N2 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...)

La fissazione dal punto di vista del batterio: tutti fissano l’azoto 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

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 all’ossigeno.Come si risolve il problema?

Fd red

Fd ox

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

spora

Le eterocisti si formano quando manca azoto.Hanno parete molto spessa che limita scambiManca PSII ( non evolvono ossigeno)Anabaena vive in simbiosi con felce Azolla

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

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 PO2)

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

Ulteriore trucco: ridurre PO2 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

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.

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

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

rhizosphereFlavonoidsnod-geneInducers

(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.

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.

Nod factors inducono allungamento pelo radicale

INFEZIONE CONTROLLATA

allungamento pelo radicale

curvatura pelo radicale

root hair beginning to curl

Rhizobiumcells

Nod factors inducono degradazione parete cellulareSi 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

Cells de-differentiate & divide nodule primordiumReady to receive bacteria from infection thread

Controllo ormonaledella crescita:Coinvolti auxina, gibberellina ed etilene

La formazione del primordio del nodulo avviene lontano dall’epidermide

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

Nodule structure and infection in wild type and Fix− mutants

Wild type

I, meristemcentral 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

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 l’infezione dei batteri si utilizzano batteri con la β-galactosidasi

Il processo è controllato da molti geni

Metabolismi accoppiati

glutamina asparagina

alla pianta

legume

rhizobia

Fixed nitrogen(ammonia)

Fixed carbon(malate, sucrose)

N2

Leghemoglobin

O2

malato

4O2

Krebs

NADH

1 N2

8 Ferredoxred

8H2016 ATP

nitrogenasi

2 NH3 + H2

48

Bacterioid metabolism

La respirazione aiuta a mantenere bassa la pO2

L’ossidasi terminale ha un’altissima affinità per O2

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

Receptor-like kinasesTransmembrane 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

Strong similarities between NOD factor and chitin sensing.

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

Myc factors: a mixture of sulphated and non-sulphated simple lipochitooligosaccharides (LCOs)

Proposed chemical structures of two major Myc-LCOs

General Myc-LCO structure

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

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

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

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.

PLANT GROWTH REGULATORSPLANT GROWTH REGULATORS

1. Endogenous1. Endogenousa. Substance produced by a plant that affects the pattern of growth and a. Substance produced by a plant that affects the pattern of growth and development.development.b. Production by the plant is regulated by the environment.b. Production by the plant is regulated by the environment.

2. Exogenous2. Exogenousa. Substance applied to the plant that alters growth and development in a. Substance applied to the plant that alters growth and development in the same way that endogenous substances do.the same way that endogenous substances do.b. May be the same or different chemically from the endogenous b. May be the same or different chemically from the endogenous substancesubstance

HormoneHormonea. Substance that acts in very low concentration (micro-molar or less)a. Substance that acts in very low concentration (micro-molar or less)b. Produced in one part of plant and act in another (translocatable)b. Produced in one part of plant and act in another (translocatable)c. Has the same response in many different plant speciesc. Has the same response in many different plant species

PrimaryPrimary 1. Auxins1. Auxins 2. Cytokinins2. Cytokinins 3. Gibberellins3. Gibberellins 4. Abscisic Acid4. Abscisic Acid 5. Ethylene5. Ethylene

Secondary Secondary - newly discovered- newly discovered 1. Jasmonic Acid1. Jasmonic Acid 2. Brassinosteroids2. Brassinosteroids 3. Juglone3. Juglone 4. Salicylic Acid4. Salicylic Acid 5. Polyamines5. Polyamines

OthersOthers - not yet confirmed or understood - 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)

A Survey of Plant Hormones

L’ormone AUXINA

Triptofano

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

Materiale sperimentale: coleottile

Auxin Discovery

Auxin Discovery

Il segnale è un fattore diffusibile che si muove verso la base del coleottile

Test biologico quantitativo (permette una misura della quantità dell’auxina)Test biologico quantitativo (permette una misura della quantità dell’auxina)

Growth and morphogenesis of root in A. thaliana.

Ben Scheres’ group:Molecular Genetics Group, Department of Biology, Utrecht University

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

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)

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)

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

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

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.

BibliografiaDixon & 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.

Infection strategies

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