2 In vista dell’esame...Lezioni in ppt disponibili ma suggerisco di utilizzare per lo studio prima testi scrittiProgramma & letteratura originaleScritto: 2 h (insieme con il prof. Bertoni)7 o 8 appelli ( )Appello subtio dopo PasquaSegnalate 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)Fitodepurazione: descrivere i vari tipi di fitodepurazione con almeno un esempio di approccio biotecnologico nel dettaglio (5 punti)
4 PROGRAMMALe piante coltivate e la sindrome da domesticazione: shattering e dormienzaRischi e benefici ambientali delle piante transgeniche in paragone a quelle convenzionali.Convenzione di Rio, Protocollo di Cartagena e normativa sulle piante create tramite ingegneria geneticaPiante 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’N2Haber 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 / yrFritz Haber( ) 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.
7 Quali batteri fissano l’azoto? Asymbiotic nitrogen fixersGroups containing symbiotic fixersKneip (2007) Nitrogen fixation in eukaryotes – New models for symbiosis BMC Evolutionary Biology 2007, 7:55.
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, AzotobacterSymbiotic fixersRhizobium in free living state
9 La fissazione dal punto di vista del batterio: tutti fissano l’azoto per mezzo della nitrogenasi 4 ATP required per pair of electrons transferredThe nitrogenase complex is comprised of two main functional subunits: dinitrogenase reductase (azoferredoxin) and dinitrogenase (molybdoferredoxin)
10 La reazione della nitrogenasi è sensibile all’ossigeno. Fe and MoFe proteins of A. vinelandii nitrogenaseLa reazione della nitrogenasi è sensibile all’ossigeno.Come si risolve il problema?Fd redFd oxNitrogenase Fe protein cycleDixon & Kahn (2004) Genetic regulation of biological nitrogen fixation. Nature Reviews Microbiology 2,
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 spora Una strategia per evitare l’inattivazione delle nitrogenasi: confinare la reazione in una cellula non ossigenicasporaLe eterocisti si formano quando manca azoto.Hanno parete molto spessa che limita scambiManca 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 and the development of a specialised organ, the root nodule.
14 Genetic regulation of biological nitrogen fixation Ray Dixon & Daniel KahnNature Reviews Microbiology 2, (August 2004)The nitrogen-fixing nodule hosts symbiotic Rhizobium bacteroids, which function as specialized nitrogen fixing organelles that exchange fixed nitrogen for photosynthates. A physiological paradox arises from the aerobic requirements of bacteroid metabolism compared with the oxygen sensitivity of nitrogenase in the absence of a dedicated bacterial protective system. Protection against oxygen is provided by the nodule environment through a cortical diffusion barrier so that the main route of oxygen diffusion is through the nodule apex, which generates a longitudinal oxygen gradient (see the figure of a starch-stained alfalfa nodule; scale bar represents 200 m). As a result, the free oxygen concentration drops to less than 50 nM in the central nitrogen-fixing zone containing Rhizobium bacteroids. Oxygen diffusion is facilitated in the central zone by a high concentration of leghaemoglobin, and bacteroid respiration is made possible by the induction of a high affinity cbb3 oxidase. Therefore, Rhizobium bacteroids fix nitrogen in a microaerobic, nitrogen-rich environment, which explains why oxygen, not nitrogen, is the main physiological regulatory factor for nif gene induction during symbiosis.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 leghaemoglobinReversible 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 LbRNAi plants grew normally when fertilized with mineral nitrogen wild-typeTen-week-old rhizobia-inoculated plants grown in sand without mineral nitrogenwild-typeindividuals of two LbRNAi linesPlants grown in grown in soil with nitrogen fertilizerLbRNAiFigure 3. Symbiotic and Nonsymbiotic Phenotypes of LbRNAi Lines(A) Typical nodules of wild-type and LbRNAi plants 14 days after inoculation with rhizobia. The top two images show whole and sectioned wild-type nodules, respectively, and the bottom pair depicts the same for LbRNAi line 3-2. Note the absence of leghemoglobin pigmentation in the LbRNAi nodule but the similar extent of dark-staining, infected cells in the central zone of the nodule section. The scale bars indicate 1 mm.(B) Ten-week-old rhizobia-inoculated plants. Three individuals of two independent LbRNAi lines (2-1, 2-2, 2-3 and 3-1, 3-2, 3-3) are compared with a typical wild-type control (far left). Plants were grown in sand without added mineral nitrogen.(C) Ten-week-old noninoculated plants grown in soil with nitrogen fertilizer. Two LbRNAi individuals are compared to a typical wild-type plant (left).LbRNAi plants grew normally when fertilized with mineral nitrogenNodules 14 days after inoculation with rhizobiaOtt T. et al., (2005) Curr Biol. 15:531-5.
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 controlsLbRNAi linesWild-typesurfacecenter of nodulesIncrease in nodule free oxygen, loss of bacterial nitrogenase protein, and absence of SNFOtt T. et al., (2005) Curr Biol. 15:531-5.
19 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.inducerinhibitorsFlavonoidsnod-geneInducers(produced by plants)Nod-factor(produced by bacteria)rhizosphereRhizobium
20 a lipo-oligosaccharide Nod Factor:a lipo-oligosaccharideNFs 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 M) but have no effect on non-host species
21 INFEZIONE CONTROLLATA Nod factors inducono allungamento pelo radicaleallungamento pelo radicalecurvatura pelo radicale
23 Nod factors: sono specie specifici (sia del batterio che della pianta) Nod factors inducono degradazione parete cellulareSi forma il tubetto infettivo per invaginazione della PMdegradation of cell wallinfection threadNod factors: sono specie specifici (sia del batterio che della pianta)
24 Cells de-differentiate & divide nodule primordium Ready to receive bacteria from infection threadControllo ormonaledella crescita:Coinvolti auxina, gibberellina ed etileneLa formazione del primordio del nodulo avviene lontano dall’epidermide
25 Medicago truncatula-Sinorhizobium meliloti interaction Il nodulo matura: forma connessioni vascolari ed esclude O2I batteri diventano batterioidi (10 v. più grandi) e iniziano a fissare N2pp4e-fig jpgIl processo è stato studiato con mutanti incapaci di fissare N2 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 I, meristemcentral area of the nodulessymbiotic cells with intracellular bacteria200 µmWild typeIII: nitrogen fixationB: differentiated cells that do not fix nitrogen(Left panels) Tissue organisation of nodules including a corresponding schematic illustration. Tissues distinguishable in the nodule are I, meristem; I* in (E), primordium-like structure with dividing cells; II, infection and differentiation zone; II* in (D), zone of infection without cellular differentiation; III, nitrogen fixation zone; III* in (B), zone with fully differentiated cells that, however, do not fix nitrogen; III** in (C), zone with differentiated plant cells in which bacteroids are not differentiated; IV, zone of symbiotic cell senescence; root and nodule cortical tissues are in grey. Bars equal 200 µm. (Middle panels) Enlargement of the central area of the nodules showing the presence or absence of differentiated symbiotic cells. Bars equal 50 µm. (Right panels) Images of symbiotic cells showing the structure of the intracellular bacteria (A–C) or the absence of differentiation and bacteria in the cells in the central area of the nodule (D,E). Bars equal 50 µm.C: differentiated plant cells with undifferentiated bacteroids50 µm50 µmSemi-thin longitudinal sections stained with toluidine blue
27 Il processo è controllato da molti geni Per visualizzare meglio l’infezione dei batteri si utilizzano batteri con la β-galactosidasiIl processo è controllato da molti geniPlant 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 Bacterioid metabolism malatoLeghemoglobinKrebsLa respirazione aiuta a mantenere bassa la pO2NADH844O2O28 FerredoxredL’ossidasi terminale ha un’altissima affinità per O216 ATP8H20nitrogenasiN21 N22 NH3 + H2
30 Signaling pathways for infection and organogenesis are known What are the molecular players involved in nodule formation?NOD factor sensing by Receptor-Like KinasesFLS2 (Flagellin-insensitive 2), a leucine rich repeat (LRR) receptor-like kinase similar to SYMRKFigure 1 A schematic representing the genetic pathways involved in the activation of nodule organogenesis and bacterial infection. Epidermal cells are capable of perceiving Nod factor (NF) through receptor-like kinases (NFR1/NFR5) that activate calcium oscillations via a suite of proteins. Perception of the calcium oscillations involves the calcium-activated kinase CCaMK, which functions with transcription factors (such as NIN and ERN1) to activate gene expression. This signaling pathway has two outcomes: the initiation of bacterial infection at the epidermis and the promotion of cell division in the cortex via an unknown diffusible signal (dotted line). In the inner or mid-cortex, NF-induced cytokinin signaling involves the cytokinin receptor LHK1/CRE1, response regulators, and the transcription factors NSP1, NSP2, and NIN. A target for cytokinin signaling is the suppression of polar auxin transport that is sufficient to promote nodule organogenesis. Although the transcription factors have been placed upstream of the changes to polar auxin transport, they could equally lie downstream.Signaling pathways for infection and organogenesis are known
31 Receptor-like kinases Transmembrane proteins with extracellular LRRs and an intracellular kinase domainReceptor/sensorProtein kinaseRLK contain: an extracellular domain, a transmembrane domain and an intracellular protein kinase domain
32 Strong similarities between NOD factor and chitin sensing.
34 Myc factors: a mixture of sulphated and non-sulphated simple lipochitooligosaccharides (LCOs) Proposed chemical structures of two major Myc-LCOsGeneral 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 Perception of AM fungal or rhizobia-derived signals triggers early signal transduction, which is mediated by at least seven shared components. The symbiosis receptor kinase SYMRK acts upstream of the Nod factor- and Myc factor-induced calcium signatures that occur in and around the nucleus. Perinuclear calcium spiking involves the release of calcium from a storage compartment (probably the nuclear envelope) through as-yet-unidentified calcium channels. The potassium-permeable channels CASTOR and POLLUX might compensate for the resulting charge imbalance. The nucleoporins NUP85 and NUP133 are required for calcium spiking, although their mode of involvement is currently unknown. The calcium–calmodulin-dependent protein kinase (CCaMK) forms a complex with CYCLOPS, a phosphorylation substrate, within the nucleus. Together with calmodulin, this complex might decode the symbiotic calcium signatures (K. Yano and colleagues, personal communication). Upstream of the common pathway, the Nod factor receptor kinases NFR1 and NFR5 are specifically required for Nod factor perception. It is possible that similar receptors are involved in Myc factor perception. Lotus japonicus protein nomenclature is used (see Table 1 for the names of common SYM gene orthologues of other species).
37 - Arbuscular mycorrhizal fungi produce NF-like molecules 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 formationBacteria are entrapped in a curled root hairinfection threads are initiatedFigure 2 Nodulation involves the coordinated development of bacterial infection and nodule organogenesis. Cell division (indicated with dotted lines) in the inner or mid-cortex and pericycle is initiated early in the interaction between the root and rhizobial bacteria and precedes the initiation of infection events. Bacteria are entrapped in a curled root hair, and from this site infection threads (ITs) are initiated. The route of the IT is predicted by pre-infection threads that are densely cytoplasmic subdomains with aligned cytoskeleton. ITs progress into the inner cortex where the nodule primordium has formed through a series of cell divisions. From these divided cells, the nodule meristem forms.Cell divisionFurther division start the nodule primordium- Components of the NF signaling pathway are also required for mycorrhizal signaling- Arbuscular mycorrhizal fungi produce NF-like molecules
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. Endogenousa. Substance produced by a plant that affects the pattern of growth and development.b. Production by the plant is regulated by the environment.2. Exogenousa. 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 substanceHormonea. 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 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
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 dataMost remarkably: the model describes root behavior quite well!e.g. distribution of different auxin transporters (PIN proteins)
51 cytokinin acts at the boundary between the proximal meristem and the elongation zone, and is associated with the transition from cell division to cellDuring 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 meristemAuxin accumulates at the tip of the root apical meristem, and this is crucial to maintain cell division
52 Auxin and cytokinin during root meristem development Lateral root initiation and nodule formation share several characteristicsLocalized 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.Cytokinin signaling during nodulation is restricted to the pericycle and cortical cells where cell divisions occur during nodule initiationFigure 3 The roles of auxin and cytokinin during root meristem development. The root apical meristem is divided into the stem cell niche where the quiescent center resides, the proximal meristem where cell divisions occur, the elongation zone where cells expand, and the differentiation zone where the specialized structures of cells emerge. As a result of auxin transporters (PINs), auxin traverses down the root and accumulates at the tip in the region of the stem cell niche. A cycle of auxin flow is very apparent in the proximal meristem region but reduced in the elongation zone, likely through cytokinin suppression of PINs. A gradient of relative importance of cytokinin and auxin is established with high auxin at the stem cell niche and high cytokinin signaling at the transition zone between the proximal meristem and the elongation zone. This gradient is maintained at least in part through antagonisms between auxin and cytokinin that are facilitated by auxin induction of RR7 and RR15 (that suppress cytokinin signaling) and cytokinin induction of SHY2 (that inhibits PIN expression). In lateral roots, 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. These increases in cytokinin signaling block auxin transport via the suppression of PINs, and it appears that low auxin levels may be associated with the initiation of the nodule primordium. Activation of nodule organogenesis requires Nod factor (NF) recognition at the root surface, and this induces the transcription factor NIN, which may be sufficient to induce cytokinin signaling in the cortex through the upregulation of CRE1.During nodule development, localized cytokinin signaling in the root cortex is necessary and sufficient for the initiation of the nodule primordiaLocalized suppression of polar auxin transport creates a minimum of auxin which induce nodule morphogenesis
53 In conclusionWe 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 formationAs 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 BibliografiaDixon & Kahn (2004) Genetic regulation of biological nitrogen fixation Nature Reviews Microbiology 2:Oldroyd et al., (2011) The Rules of Engagement in the Legume-Rhizobial Symbiosis. Annu. Rev. Genet. 45: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: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.