Plant Nutrition 2: Macronutrients (N, P, K, S, Mg

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1 Plant Nutrition 2: Macronutrients (N, P, K, S, Mg and Ca)

2 Plant Nutrition 3: Micronutrients and Metals D-block metals Metals Metalloids Non-metals Essential micronutrients Non-essential toxic elements (examples) Essential for animals, beneficial for plants

3 Plants assimilate mineral nutrients mainly as cations or MACRONUTRIENTS μmol / g Element (dry wt) 250 Potassium (K) K + Assimilated form 1000 Nitrogen (N) NO 3, NH Phosphorus HPO 2 4, (P) H 2 PO 4 30 Sulfur (S) SO Magnesium (Mg) Mg 2+ anions MICRONUTRIENTS μmol / g Element (dry wt) Assimilated form 2 Iron (Fe) Fe 3+, Fe Nickel (Ni) Ni + 1 Manganese Mn 2+ (Mn) 0.1 Copper (Cu) Cu Molybdenum (Mo) MoO Calcium (Ca) Ca 2+ 2 Boron (B) H 3BO 3 Charged ions require transport proteins to cross membranes 3 Chlorine (Cl) Cl 0.3 Zinc (Zn) Zn 2+ See Taiz, L. and Zeiger, E. (2010) Plant Physiology. Sinauer Associates; Marschner, P. (2012) Mineral Nutrition of Higher Plants. Academic Press, London

4 Aφομοίωσηανόργανωνθρεπτικών στοιχείων Άζωτο (αμινοξέα, πρωτεϊνες, νουκλεϊκά οξέα, χλωροφύλλες κ.α.) Φωσφόρος (φωσφορυλίωση ενώσεων, φωσφολιπίδια, ATP, PPi κ.α) Κάλιο (τροπισμοί, ωσμωτική ρύθμιση, ενεργοποίηση ενζύμων κ.α.) Θείο (αμινοξέα, συνένζυμο Α, θειαμίνη, βιοτίνη κ.α.) Ασβέστιο (συστατικό κυτταρικής πλάκας, ρύθμιση αύξησης και ανάπτυξης) Μαγνήσιο (χλωροφύλλη, ρ φ ενεργοποίηση η κινασών κ.α.) ) Χλώριο (αυξίνη, κυτταροδιαιρέσεις, ώσμωση, φωτόλυση κ.α.) Σίδηρος (χλωροφύλλη, καταλυτική δράση, κυτοχρώματα, κ.α.) Χαλκός (κυτοχρωμική οξειδάση, πλαστοκυανίνη κ.α.) Ψευδάργυρος (ενεργότητα ενζύμων π.χ. ινδολυλοξικού οξέος κ.α.) Μολυβδαίνιο (αναγωγή νιτρικών, καταβολισμός πουρινών, κ.α.) Νικέλιο (ουρεάση) Μαγγάνιο (φωτόλυση, μεμβράνες χλωροπλαστών) Βόριο (σύνθεση DNA /RNA κ.α.)

5 Vascular plants assimilate mineral nutrients mostly via roots By increasing surface area for absorption, root hairs functionally resemble microvilli of an animal s intestinal epithelium Membrane transporters facilitate nutrient uptake Barberon, M. and Geldner, N. (2014). Radial transport of nutrients: the plant root as a polarized epithelium. Plant Physiol. 166:

6 Roots have several adaptations to enhance nutrient capture Biochemical responses Developmental responses Fungal symbiotic partners Prokaryotic symbiotic partners Schmidt, S., Raven, J.A. and Paungfoo-Lonhienne, C. (2013). The mixotrophic nature of photosynthetic plants. Funct. Plant Biol. 40: by permission of CSIRO publishing.

7 P Symbioses Nutrient uptake, assimilation Root exudates N N and utilization involve many P NH 3 Nutrient uptake efficiency Root system architecture processes Transporters and pumps Intercellular transport efficiency Nutrient utilization efficiency X R-X Assimilation and remobilization efficiency Regulatory and control networks Rhizosphere microbiota

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9 Αφομοίωση αζώτου

10 Ο βιογεωχημικός κύκλος του αζώτου στη φύση Βιομηχανική δέσμευση Μέθοδος Haber-Bosch Βιολογική δέσμευση Ατμοσφαιρική δέσμευση Φωτοχημικές αντιδράσει Αστραπές Βροχή (ΗΝΟ3)

11 Nitrogen is an essential nutrient Alanine and other amino acids contain nitrogen Nitrogen is the 4 th most abundant element in living organisms. It is a component of amino acids and proteins, nucleic acids (DNA and RNA), chlorophyll, hormones, and secondary metabolites. Chlorophyll a Adenine and other nucleoside bases contain nitrogen From: Buchanan, B.B., Gruissem, W. and Jones, R.L. (2000) Biochemistry and Molecular Biology of Plants. American Society of Plant Physiologists.

12 Nitrogen can be found in many Species Name Oidti Oxidation State R NH 2 Organic nitrogen, urea 3 NH 3, NH + 4 Ammonia, 3 ammonium ion N 2 Nitrogen 0 N 2 O Nitrous oxide +1 NO Nitric oxide +2 HNO 2, NO 2 Nitrous acid, +3 nitrite ion NO 2 Nitrogen dioxide +4 HNO 3, NO 3 Nitric acid, nitrate ion +5 inorganic forms NO - 3 Nitrate reduction NO 2 - NO 2 - Nitrificationifi ti NO N 2 O Aerobic reactions Anaerobic reactions N 2 NH 3 Nitrogen fixation Adapted from Robertson, G.P. and Vitousek, P.M. (2009). Nitrogen in agriculture: Balancing the cost of an essential resource. Annu. Rev. Environ. Res. 34:

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14 Nitrogen is abundant but unavailable Atmospheric composition N N Other gases 1% O 2 21% N 2 78% N 2 In the atmosphere, nitrogen exists as dinitrogen gas N 2, an unusually inert molecule with a triple bond holding the two atoms together

15 N N Nitrogen must be fixed (reduced) and this triple bound broken so that N becomes biologically available. N 2 3 H 2 The nitrogen-fixation reaction demands a high input of energy. 2 NH 3 (ammonia)

16 Nitrogen fixation occurs through biological and non-biological processes Source Lightning Biological N-fixation (terrestrial systems) Biological N-fixation (marine systems) N fertilizer synthesis Fossil fuel combustion Amount of N fixed <10 Tg/year Tg/year Tg/year 80 Tg/year >20 Tg/year From: Buchanan, B.B., Gruissem, W. and Jones, R.L. (2000) Biochemistry and Molecular Biology of Plants. American Society of Plant Physiologists.

17 The Haber-Bosch process of industrial nitrogen fixation N H 2 2 NH 3 ~500 Atmospheres ~600 C Catalyzed reaction Fritz Haber Carl Bosch Very energetically expensive!

18 Nitrogen fixed by the Haber-Bosch process sustains half the human population World population 2 Population sustained by 1 industrially fixed nitrogen Plant sequestered N is the source, directly and indirectly, of all human nutritional N Lack of N availability in the developing world limits crop production, but overuse of N fertilizers poses environmental threats Source: Erisman, J.W., Sutton, M.A., Galloway, J., Klimont, Z., and Winiwarter, W. (2008). How a century of ammonia synthesis changed the world. Nature Geosci 1:

19 Run off from nitrogenous fertilizers is a major pollutant Fertilizer application produces greenhouse gases (e.g. nitrous oxide) and pollutes waterways Algal bloom due to nitrogen run-off in the Gulf of Mexico The surface of a pond showing algal overgrowth Image source: Lamiot Sources: IFA and UN; Photo courtesy of NASA/Goddard Space Flight Center Scientific Visualization Studio

20 Fixing nitrogen to fertilize plants accounts for ~2% of global energy use CO 2

21 How do plants optimize their uptake and utilization of How is inorganic nitrogen assimilated into organic molecules? How is nitrogen taken up into the plant? nitrogen? How do plants sense local soil nitrogen levels and plant nitrogen status? How do plants respond to nitrogen deficit? How do they maximize uptake through their roots? How do plants remobilize nitrogen to optimize N- utilization?

22 Nitrogen metabolism: Uptake, assimilation and remobilization Uptake Remobilization Amino acid recycling, photore spiration Glutamate Assimilation Carbon pools TCA cycle 2-oxoglutarate NH 4 + Nitrate reductase NO 3 - NO 3 - R-NH 2 NH + 4 Nitrite reductase NO - 2 Assimilation NH + 4 Glutamine synthetase (GS) Glutamine Glutamine-2- oxoglutarate aminotransferase (GOGAT) Glutamate Incorporation into amino acids and other nitrogencontaining compounds N 2 Adapted from Xu, G., Fan, X. and Miller, A.J. (2012). Plant nitrogen assimilation and use efficiency. Annu. Rev. Plant Biol. 63:

23 Most plants take up most of their nitrogen as nitrate NO - 3 Many prokaryotes oxidize NH 4+, so soil NH 4+ levels are often low Plants use energy to reduce NO 3- for assimilation into organic compounds Energy released Energy released Energy consumed Energy consumed NH 4 + NO 2 - NO 3 - Nitrification by nitrifying prokaryotes NO 3 - Nitrate reductase NO 2 - NH 4 + Nitrite reductase R-NH 3 Plant preferences for NH 4+ vs NO 3- vary by species, other metabolic processes, temperature, water, soil ph etc. See Li, B., Li, G., Kronzucker, H.J., Baluška, F. and Shi, W. (2014). Ammonium stress in Arabidopsis: signaling, genetic loci, and physiological targets. Trends Plant Sci. 19: ; Britto, D.T. and Kronzucker, H.J. (2013). Ecological significance and complexity of N-source preference in plants. Ann. Bot. 112:

24 Πρόσληψη και αφομοίωση του αζώτου από τα φυτά (Α) αφομοίωση νιτρικών ΝΟ3-1. Αποθήκευση στο χυμοτόπιο 2. Αναγωγή σε νιτρώδη-αμμωνιακά 3. Μεταφορά μέσω του ξυλώματος στο υπεργειο τμήμα 3 1 2

25 Plants have specific transporters for NO - + 3, NH 4 and other N forms HATS = high affinity transporters LATS = low affinity transporters Nacry, P., Bouguyon, E. and Gojon, A. (2013). Nitrogen acquisition by roots: physiological and developmental mechanisms ensuring plant adaptation to a fluctuating resource. Plant Soil. 370: 1-29, With kind permission from Springer Science and Business Media

26 Primary N assimilation: NO 3- is reduced to NH 4+ prior to Uptake assimilation Glutamate NH 4 + NO 3 - NO 3 - NH + 4 Nitrite reductase NO - Nitrate 2 reductase Glutamine Guta synthetase (GS) Assimilation into organic compounds Glutamine All other N- containing i compounds R-NH 3

27 Αναγωγή νιτρικών ιόντων σε νιτρώδη Νιτρική αναγωγάση (nitrate reductase-nr) Δ ιμερές ένζυμο με δύο προσθετικές ομάδες (FAD και αίμη) Συμπαράγοντας μολυβδαινίου με πτερίνη Στο κυτταρόπλασμα Τρια ισοένζυμα

28 Αναγωγή νιτρωδών σε αμμωνιακά ιόντα ΝΟ Fd αν + 8Η + + 6e - NH Fd οξ. + 2Η 2 Ο Aναγωγάση νιτρωδών (nitrite reductase-νir) Δυο προσθετικές ομάδες (αίμη και σύμπλοκο σιδήρου-θείου) Διαφορετικά ισοένζυμα στους χλωροπλάστες και στα πλαστίδια

29 Ρύθμιση της αναγωγής νιτρικών ιόντων Ρύθμιση των συστημάτων πρόσληψης (συμ-μεταφορέων) Ρύθμιση των ΝR και NiR Ρύθμιση σε μεταγραφικό, μετα-μεταγραφικό, και μετα-μεταφραστικό επίπεδο (+): νιτρικά ιόντα,, φως, διαθεσιμότητα σακχάρων,, κυτοκινίνη (-): γλουταμίνη, ασπαραγίνη (τελικά προϊόντα), σκοτάδι, συνθήκες καταπόνησης, χαμηλή συγκέντρωση CO2,

30 Πρόσληψη και αφομοίωση του αζώτου από τα φυτά (B) αφομοίωση αμμωνίας 1. Ενσωμάτωση σε αζωτούχα βιομόρια (αμινοξέα μ ξ κυρίως) 2. Μεταφορά μέσω του ξυλώματος στο υπεργειο τμήμα Πηγές αμμωνιακών στα φυτά Πρόσληψη από τις ρίζες Αναγωγή νιτρικών Συμβιωτική αζωτοδέσμευση Απαμινώσεις Φωτοαναπνοή Μεταβολισμός φαινυλοπροπανοειδών

31 Rebmobilization of N occurs during senescence and Each N atom may cycle through GS many times as amino acids are recycled during growth and senescence and released due to photorespiration photorespiration assimilation remobilization Amino acids Leaves Roots, Cotyledons Amino acids remobilization Glutamate Fdx-GOGAT Glutamate NADH-GOGAT Glutamate Chloroplast localized GS2 Glutamine Glutamate Cytosolic GS1 NH + AA 4 NH + 4 catabolism Assimilation Glutamine Assimilation Photo- respiration assimilation uptake Avice, J.-C. and Etienne, P. (2014). Leaf senescence and nitrogen remobilization efficiency in oilseed rape (Brassica napus L.). J. Exp. Bot. 65: by permission of Oxford University Press.

32 φωτοαναπνοή

33 H αμμωνία (αμμωνιακά ιόντα) είναι τοξική για τους οργανισμούς

34 Βιοσύνθεση αμινοξέων

35 Γλουταμινική Συνθετάση υθεάση Δύο ισοένζυμα, κυτταροπλασματική GS1 και πλαστιδιακή GS2 GS1: σε βλαστάνοντα σπέρματα, στο αγωγό γ σύστημα για μεταφορά αζώτου GS2: στη ρίζα για τοπική κατανάλωση, στα φύλλα κατά τη φωτοαναπνοή- επίδραση από φως και διαθέσιμους υδατάνθρακες

36 Συνθετάση του γλουταμινικού Δύο ισοένζυμα, με δότες e- είτε NADH (πλαστίδια μη φωτοσυνθετικών ιστών,π.χ. ρίζα, αγωγό σύστημα φύλλων) είτε ανηγμένη φερρεδοξίνη (χλωροπλάστες, θετική ρύθμιση από το φως)

37 GS/GOGAT assimilates inorganic nitrogen into organic Remobilization Amino acid recycling, photorespiration ti molecules Glutamate Assimilation Carbon pools TCA cycle 2-oxoglutarate Uptake NH 4 + Glutamine-2- oxoglutarate oguta ate aminotransferase (GOGAT) Glutamate Glutamine synthetase (GS) Glutamine Incorporation into amino acids and other nitrogencontaining compounds

38 Αφυδρογονάση του γλουταμινικού Δύο ισοένζυμα, μιτοχονδριακή (ΝADH) και χλωροπλαστική (ΝΑDPH)

39 Ενεργοποιείται από συνθήκες έλλειψης ενέργειας Ν:C= 2:4 Μεταφορά και αποθήκευση αζώτου

40 Το ενεργειακό κόστος της φωτοαφομοίωσης είναι περίπου το 25% του συνολικού τόσο στις ρίζες όσο και στο υπέργειο τμήμα του φυτού

41 Α ό ύ ύ θ ί Από πού προκύπτουν οι ανθρακικοί σκελετοί στους οποίους ενσωματώνεται η αμμωνία;

42 Αλλά και

43 Regulation: Nitrogen sensing, signaling and deficit responses NITROGEN DEFICIT Increase uptake Activation of some NO 3- and NH 4+ transporters Preferential growth of root relative to shoot Metabolic adaptations to low-n Decreased accumulation of N-rich chlorophyll Increased accumulation N-free anthocyanins Smaller pools of N-containing compounds (amino acids) Larger pools of N-free compounds (starches, organic acids) Accelerated senescence and nitrogen remobilization See for example Scheible, W.-R., et al and Stitt, M. (2004). Genome-wide reprogramming of primary and secondary metabolism, protein synthesis, cellular growth processes, and the regulatory infrastructure of Arabidopsis in response to nitrogen. Plant Physiol. 136: ; Krapp, A. et al and Daniel-Vedele, F. (2011). Arabidopsis roots and shoots show distinct temporal adaptation patterns toward nitrogen starvation. Plant Physiol. 157: Schlüter, U., et al. and Sonnewald, U. (2012). Maize source leaf adaptation to nitrogen deficiency affects not only nitrogen and carbon metabolism but also control of phosphate homeostasis. Plant Physiol. 160: Amiour, N. et al and Hirel, B. (2012). The use of metabolomics integrated with transcriptomic and proteomic studies for identifying key steps involved in the control of nitrogen metabolism in crops such as maize. J. Exp. Bot. 63: Balazadeh, S., et al. and Mueller-Roeber, B. (2014). Reversal of senescence by N resupply to N-starved Arabidopsis thaliana: transcriptomic and metabolomic consequences. J. Exp. Bot. 63:

44 Roots respond to local and systemic nitrogen availability When nitrogen is abundant, plants allocate less biomass to their roots When nitrogen distribution is patchy, roots proliferate in the nutrient rich patches Reprinted by permission from Wiley from Drew M C (1975) Comparison of the effects of a localised supply of phosphate nitrate and ammonium and potassium on the growth of the seminal Reprinted by permission from Wiley from Drew, M.C. (1975). Comparison of the effects of a localised supply of phosphate, nitrate and ammonium and potassium on the growth of the seminal root system, and the shoot, in barley. New Phytol. 75: Reprinted from Bouguyon, E., Gojon, A. and Nacry, P. (2012). Nitrate sensing and signaling in plants. Sem. Cell Devel. Biol. 23: , with permission from Elsevier. See also Gersani, M. and Sachs, T. (1992). Development correlations between roots in heterogeneous environments. Plant Cell Environ. 15:

45 The split-root system separates local and systemic signals All plants split with ½ root system in each of two chambers C.NO3 plants Both chambers contain KNO 3 (local and systemic signals indicate NO 3 available) Sp.NO3 roots experience locally high h NO 3- but also N-deficiency signals derived from Sp.KCl roots Sp.KCl roots Experience locally deficient i NO - 3 conditions but also N-sufficient signals from Sp.NO3 roots C.KCl plants Both chambers contain KCl (local and systemic signals indicate NO 3- deficiency) Ruffel, S., Krouk, G., Ristova, D., Shasha, D., Birnbaum, K.D. and Coruzzi, G.M. (2011). Nitrogen economics of root foraging: Transitive closure of the nitrate cytokinin relay and distinct systemic signaling for N supply vs. demand. Proc. Natl. Acad. Sci. USA 108:

46 Evidence for a systemic signal of N-demand Signals on from root the N-replete development Signals from the N- deficient roots promote Sp.NO3 roots supress root elevated root growth in growth in Sp.KCl as Sp.NO3 as compared to compared to C.KCl roots, C.NO3, indicating that t a indicating that a response to systemic N-repletion signals response to systemic N- starvation signals Model: Systemic signals promote root growth and suppress root growth Ruffel S Krouk G Ristova D Shasha D Birnbaum K D and Coruzzi G M (2011) Nitrogen economics of root foraging: Transitive closure of the nitrate cytokinin relay and distinct systemic signaling for N Ruffel, S., Krouk, G., Ristova, D., Shasha, D., Birnbaum, K.D. and Coruzzi, G.M. (2011). Nitrogen economics of root foraging: Transitive closure of the nitrate cytokinin relay and distinct systemic signaling for N supply vs. demand. Proc. Natl. Acad. Sci. USA 108: Li, Y., Krouk, G., Coruzzi, G.M. and Ruffel, S. (2014). Finding a nitrogen niche: a systems integration of local and systemic nitrogen signalling in plants. J. Exp. Bot. 65: by permission of Oxford University Press.

47 CHL1/NRT1.1/NPF6.3 is a nitrate transceptor (sensor) In wild-type plants (Ws), lateral root growth is stimulated in High Nitrate (HN) Lateral roots of transceptor mutants (chl1-10) fail to respond to the HN environment Transceptor mutants (chl1-5) also show abnormal transcriptional responses to nitrate WT chl1-5 Remans, T., et al. and Gojon, A. (2006). The Arabidopsis NRT1.1 transporter participates in the signaling pathway triggering root colonization of nitrate-rich patches. Proc. Natl. Acad. Sci. 103: by the National Academy of Sciences; Krouk, G., et al. and Gojon, A. (2010). Nitrate-regulated auxin transport by NRT1.1 defines a mechanism for nutrient sensing in plants. Devel. Cell. 18: with permission from Elsevier.

48 Model for transceptor action: NO - 3 competes for auxin When NO 3- is low, NPF6.3 transports auxin away from the root tip and growth is inhibited transport When NO 3- is high, auxin transport through NPF6.3 is suppressed and growth is promoted NPF6.3 Auxin NPF6.3 Auxin NO 3 - NO 3 - Beeckman T and Friml J (2010) Nitrate contra auxin: Nutrient sensing by roots Devel Cell 18: with permission from Elsevier See also Krouk G et al and Gojon A (2010) Nitrate regulated auxin transport by NRT1 1 defines a Beeckman, T. and Friml, J. (2010). Nitrate contra auxin: Nutrient sensing by roots. Devel. Cell. 18: with permission from Elsevier. See also Krouk, G., et al and Gojon, A. (2010). Nitrate-regulated auxin transport by NRT1.1 defines a mechanism for nutrient sensing in plants. Devel. Cell. 18: ; Mounier, E., et al and Nacry, P. (2014). Auxin-mediated nitrate signalling by NRT1.1 participates in the adaptive response of Arabidopsis root architecture to the spatial heterogeneity of nitrate availability. Plant Cell Environ. 37: ; Forde, B.G. (2014). Nitrogen signalling pathways shaping root system architecture: an update. Curr. Opin. Plant Biol. 21:

49 Biological nitrogen fixation 3 H 2 16 ATP N 2 Nitrogenase Many prokaryotes can fix nitrogen using an enzyme called nitrogenase. This process uses a great deal of cellular energy, ATP 2NH 3

50 Biological nitrogen fixation Many nitrogen-fixing bacteria are freeliving, others form associations with plants or fungi Cyanobacteria are photosynthetic nitrogen-fixing bacteria that can be free-living or mutualistic Azolla ferns, which fix nitrogen with their symbiotic partner Anabaena, are used as green fertilizer in rice paddies Microscope by Ben; Culture Collection of Autotrophic Organisms ( CCALA ) Institute of Botany, Academy of Sciences of the Czech Republic,; Azolla courtesy of IRRI

51 The presence of nif nitrogen fixation genes into very diverse prokaryotes is thought to have originated by horizontal gene transfer Diazotrophs (nitrogen-fixers) Tight gene linkage are indicated in red. Dashed facilitates horizontal lines indicate lineages with nif gene transfer of genes not known to fix nitrogen these complex traits Raymond, J., Siefert, J.L., Staples, C.R. and Blankenship, R.E. (2004). The natural history of nitrogen fixation. Mol. Biol. Evol. 21: by permission of Oxford University Press.

52 Symbiotic nitrogen fixation Some nitrogen-fixing bacteria form an intimate partnership with plants in the form of nitrogenfixing nodules Legumes with symbiotic rhizobia Actinorhizal plants like alder with symbiotic Frankia bacteria

53 Συμβιωτική δέσμευση του αζώτου

54 Parasponia Legumes Nodulating gplants form a single clade within the angiosperms (Rosids I). Actinorhizal plants are indicated by purple arrows, and plants nodulated d by rhizobia with red arrows Soltis, D.E., Soltis, P.S., Morgan, D.R., Swensen, S.M., Mullin, B.C., Dowd, J.M. and Martin, P.G. (1995). Chloroplast gene sequence data suggest a single origin of the predisposition for symbiotic nitrogen fixation in angiosperms. Proc. Natl. Acad. Sci. USA. 92: Copyright (1995) National Academy of Sciences, USA

55 Rhizobia are a large diverse group of bacteria that nodulate legumes Rhizobia (bold) distribution within Proteobacteria subdivisions Reprinted by permission from Macmillan Publishers Ltd: Moulin, L., Munive, A., Dreyfus, B. and Boivin-Masson, C. (2001). Nodulation of legumes by members of the β-subclass of Proteobacteria. Nature. 411: , copyright 2001.

56 Steps in nodule development Generalized steps in the formation of a nodule Nodule expressing leghemoglobin Communication Rhizobia Root hair curling Infection thread formation Bacteroid N 2 Plant root Cell division in root NH + 4 Adapted from Gibson, K.E., Kobayashi, H., and Walker, G.C. (2008). Molecular determinants of a symbiotic chronic infection. Annu. Rev. Genet. 42:

57 Communication: Flavonoids and Nod factors Rhizobia 1. The plant root produces specific flavonoids that attract t rhizobia Plant cell 3. The plant prepares to form a symbiotic nodule structure 2. Most rhizobia produce Nod factors, identifying them as appropriate symbionts

58 Έκφραση Νοd (nodulin) γονιδίων νημάτιο μόλυνσης καταβολή (primordium) φυματίου

59 Nod παράγοντες: λιπο-ολιγοσακχαρίτες μόρια σηματοδότησης για τη δημιουργία του φυματίου nod (nodulation) γονίδια (nodabc, D, E, F, L)

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61 Βιολογική Δέσμευση (Καθήλωση) του Αζώτου Νιτρογενάση: Δινιτρογενάση αναγωγάση και Δινιτρογενάση Δινιτρογενάση αναγωγάση (dinitrogenase reductase): ομοδιμερές με Fe Δινιτρογενάση (dinitrogenase): ετεροτετραμερές με Fe, Mo και συμπαράγοντα Fe Mo Co

62 Nitrogenase is deactivated by molecular oxygen Question: How can the energy demands of nitrogenase be met while protecting it from oxygen? Oxidative phosphorylation produces ATP efficiently but requires oxygen. From: Buchanan, B.B., Gruissem, W. and Jones, R.L. (2000) Biochemistry and Molecular Biology of Plants. American Society of Plant Physiologists.

63 Aζωτοδεσμευτικο κυανοβακτήριο Anabaena

64 Free-living diazotrophs use behavioural, physical and biochemical strategies heterocysts Heterocysts in some cyanobacteria are thick walled, anaerobic cells that fix nitrogen and are not photosynthetic. They are supported metabolically by the associated photosynthetic cells In some diazotrophs, the FeSII protein protects nitrogenase transiently and reversibly when oxygen levels are high Behavioural: Avoid oxygen (anaerobic bacteria) Aerotaxis Physical: Oxygen diffusion barriers Spatial separation from photosynthesis Biochemical: De novo synthesis of nitrogenase High respiration rates to consume O 2 Conformational protection, ti FeSII protein Gallon, J.R. (1992). Reconciling the incompatible: N 2 fixation and O 2. New Phytol. 122: ; Lery, L., Bitar, M., Costa, M., Rossle, S. and Bisch, P. (2010). Unraveling the molecular mechanisms of nitrogenase conformational protection against oxygen in diazotrophic bacteria. BMC Genomics. 11: S7.

65 Bacteroids need a high O 2 flux but low O 2 environment Oxidative phosphorylation requires oxygen for ATP production C 6 H 12 O 6 O 2 A low oxygen environment is maintained by an oxygen permeability barrier Leghemoglobin gives nodules their pink color A high affinity cytochrome oxidase in the bacteroid functions at low oxygen concentrations Leghemoglobin buffers oxygen and delivers it to respiring symbiotic cells From: Buchanan, B.B., Gruissem, W. and Jones, R.L. (2000) Biochemistry and Molecular Biology of Plants. American Society of Plant Physiologists.

66 Most rhizobia are unable to make an essential cofactor, homocitrate nifv encodes homocitrate synthase, which makes homocitrate, an essential cofactor for nitrogenase From: Buchanan, B.B., Gruissem, W. and Jones, R.L. (2000) Biochemistry and Molecular Biology of Plants. American Society of Plant Physiologists.

67 Diazotrophs that fix nitrogen as free-living bacteria have nifv Free-living diazotroph (e.g. cyanobacteria): nifv Homocitrate produced Nitrogenase activity genes Free-living rhizobia: No nifv No homocitrate No nitrogenase activity How do bacteroids fix nitrogen without nifv?

68 Diazotrophs that fix nitrogen as free-living bacteria have nifv genes Free-living diazotroph (e.g. cyanobacteria): The plant provides the homocitrate to the bacteroids nifv Homocitrate produced Nitrogenase activity Free-living rhizobia: No nifv No homocitrate How do bacteroids fix No nitrogenase activity nitrogen without nifv?

69 Root nodule symbiosis beyond legumes Parasponia is the only known plant genus outside the legumes that t is nodulated d by rhizobia Parasponia Legumes Actinorhizal plants are diverse and associate with a single genus of bacteria, Frankia

70 Rhizobia form nodules on the non-legume Parasponia Vascular tissue Nodule Parasponia rugosa Infection zone Spontaneous nodule formation in Parasponia carrying dominant active CCaMK stele Photo credit: Leonardo Co c/o Phytoimages; Webster, G., Poulton, P.R., Cocking, E.C. and Davey, M.R. (1995). The nodulation of micro-propagated plants of Parasponia andersonii by tropical legume rhizobia. J. Exp.Bot. 46: by permission of Oxford University Press; Op den Camp, R., Streng, A., De Mita, S., Cao, Q., Polone, E., Liu, W., Ammiraju, J.S.S., Kudrna, D., Wing, R., Untergasser, A., Bisseling, T., and Geurts, R. (2011). LysM-type mycorrhizal receptor recruited for rhizobium symbiosis in nonlegume Parasponia. Science 331: reprinted with permmission from AAAS.

71 Frankia nodulate ~200 species Casuarina equisetifolia of actinorhizal plants Myrica gale (bog myrtle) is used to treat stomach aches and repel insects Casuarina are named for Casuarius (cassowary) because their leaves resemble the bird s feathers Alder nodules Photo credits: Amy Ferriter, State of Idaho, Bugwood.org; Scott Hamlin; Rosser1954; Sten Porse.

72 Frankia are filamentous bacteria that form nitrogen-fixing vesicles Frankia showing vesicles (V) in which nitrogen-fixation takes place Frankia nodules with infected cells Photo credit: Ann Hirsch Benson, D.R. and Silvester, W.B. (1993). Biology of Frankia strains, actinomycete symbionts of actinorhizal plants. Microbiol. Mol. Biol. Rev. 57: reproduced with permission from the Photo credit: Ann Hirsch Benson, D.R. and Silvester, W.B. (1993). Biology of Frankia strains, actinomycete symbionts of actinorhizal plants. Microbiol. Mol. Biol. Rev. 57: reproduced with permission from the American Society for Microbiology. Gherbi, H., Markmann, K., Svistoonoff, S., Estevan, J., Autran, D., Giczey, G., Auguy, F., Péret, B., Laplaze, L., Franche, C., Parniske, M., and Bogusz, D. (2008). SymRK defines a common genetic basis for plant root endosymbioses with arbuscular mycorrhiza fungi, rhizobia, and Frankiabacteria. Proc. Natl. Acad. Sci. USA 105: Copyright National Academy of Sciences.

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74 Γλουταμίνη Ασπαραγίνη Πουρίνες-Ουρεϊδες (αλλαντοϊνη, αλλαντοϊκό οξύ Μεταφορά μέσω του ξυλώματος στο υπέργειο τμήμακαταβολισμός σε ΝΗ4

75 Symbiotic nitrogen fixation requires an exchange of nutrients The plant provides organic carbon for use in ATP production The bacteroid fixes N 2 to NH 3 which is exported to the plant cell The plant assimilates NH 4+ into amino acids using glutamine synthase Reprinted from Prell, J., and Poole, P. (2006). Metabolic changes of rhizobia in legume nodules. Trends Microbiol. 14: with permission from Elsevier.

76 The bacteroids depend upon the plant for amino acids uninoculated mutant Bacteria deficient in amino acid transporters don t fix nitrogen wild- type Amino acids are taken up by the bacteroid Reprinted from Prell, J., and Poole, P. (2006). Metabolic changes of rhizobia in legume nodules. Trends Microbiol. 14: with permission from Elsevier; Reprinted by permission from Macmillan Publishers Ltd: Lodwig, E.M., Hosie, A.H.F., Bourdes, A., Findlay, K., Allaway, D., Karunakaran, R., Downie, J.A., and Poole, P.S. (2003). Amino-acid cycling drives nitrogen fixation in the legume-rhizobium symbiosis. Nature 422:

77 Αφομοίωση θείου SO4 2-, SO2, H2SO4

78

79 Ενεργοποίηση των θειικών ιόντων SO Mg-ATP 5 φωσφοροθειϊκή αδενοσίνη (APS) + PPi Θειολάση του ΑTP Δύο ισομορφές: στα πλαστίδια και στο κυτόπλασμα Ανόργανη πυροσφοσφατάση

80 θειομεταφοράση Φωσφοροθειική φωσφοροανεδοσίνη φ η( (PAPS) π.χ. θειολιπίδια,γλουκοσυνολικά οξέα,μπρασινοστεροειδή, χολίνη, φλαβονόλη,πολυσακχαρίτες APS κινάση στο κυτόπλασμα

81 Aναγωγή των θειικών ιόντων Θειομεταφοράση του APS Αναγωγάση του θειοσουλφονικού σουλφονικό

82 Βιοσύνθεση κυστεϊνης Ακετυλοτρανσφεράση της σερίνης Θειολυάση της Ο-ακετυλοσερινης

83 στα πλαστίδια (χλωροπλάστες

84 Βιοσύνθεση μεθειονίνης

85 Αφομοίωση σιδήρου Fe 3+, Fe(OH) 2+, Fe(OH)3, Fe(OH)4 - στο έδαφος Έκκριση πρωτονίων για τη διαλυτοποίηση του σιδήρου Έκκριση χηλικών ενώσεων (π.χ. καφεϊκό οξύ, κιτρικό οξύ, μηλικό οξύ κ.α.) Έκκριση φυτοσιδηροφόρων (π.χ. χ αβενικό οξύ- στα σιτηρά) παρομοίως και Μn, Zn, Cu Απορρόφηση η από τις ρίζες ρζ

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87 Αφομοίωση σιδήρου Απορρόφηση από τις ρίζες Αναγωγή στην επιφάνεια των ριζών σε Fe 2+ Mεταφορά φ ρ μετά από ένωση με ενδογενείς ςχηλικούς φορείς ς( (κυρίως ρ ςκιτρικό) ) στα φύλλα και ενσωμάτωση (π.χ. δακτύλιος πορφυρίνης αίμης από τη σιδηροχηλάση) Αποθήκευση περίσσειας σε σύμπλοκα πρωτεϊνών-σιδήρου (φυτοφερριτίνη)

88 Αφομοίωση φμ ηφωσφόρουφ H2PO4 -, H2PO4 2- Έκκριση πρωτονίων για τη διαλυτοποίηση του ανόργανου P Aύξηση των ριζικών τριχιδίων και μεταβολή της αρχιτεκτονικής της ρίζας Αύξηση της έκφρασης των μεταφορέων των φωσφορικών ριζών από τη μεμβράνη της ρίζας Δημιουργία μυκορριζών Μεταφορά μέσω του φλοιώματος και ενσωμάτωση σε ADP (μιτοχονδρια, χλωροπλάστες) σε 13 1,3 διφωσφογλυκερινικό (κυτόπλασμα) ό λ κτλ.

89 Plants have difficulty taking up insoluble phosphate h Phosphorous is taken up from the soil as inorganic phosphate, HPO 3 -

90 Because of its low solubility, phosphate near the root is rapidly depleted

91 Ενδομυκόρριζα Εκτομυκόρριζα

92 Depletion Zone Mycorrhizal fungi extend farther into the soil, greatly enhancing phosphate assimilation

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94 Ποια είδη μυκόρριζας συναντούμε; MYKOΡΡΙΖΑ Ενδομυκόρριζα Εκτομυκόρριζα Δενδροειδής (Αrbuscular) Ericaceous Orchidaceous Paris Ericoid Arum Arbutoid Monotropoid

95 (Ecto) (Endo) Mycorrhizal fungi proliferate within or between cells Ectomycorrhizal fungi proliferate outside the root and between cells, endomycorrhizal fungi (including arbuscular mycorrhizal fungi) proliferate within cells ~3%of ~ 80% of land plants land plants Reprinted by permission from Macmillan Publishers Ltd. Bonfante, P., and Genre, A. (2010). Mechanisms underlying beneficial plant fungus interactions in mycorrhizal symbiosis. Nat Commun 1: 48.

96 Signaling between AM fungi and plant Fungi 2. The fungi produce a Myc factor, identifying it as a symbiont 1. The plant root produces strigolactones and other chemicals that initiate iti t branching in fungal hyphae Plant cell 3. The plant prepares to permit the fungi to penetrate its cells

97 The characterized Myc factors are lipochito-oligosaccharides Myc factors have a backbone of N-acetylglucosamine (chitin) sugar residues and a lipid moiety (LCOs) Some compounds identified as Myc factors are similar to rhizobial Nod factors Reprinted by permission from Macmillan Publishers Ltd. Maillet, F., Poinsot, V., Andre, O., Puech-Pages, V., Haouy, A., Gueunier, M., Cromer, L., Giraudet, D., Formey, D., Niebel, A., Martinez, E.A., Driguez, H., Becard, G. and Denarie, J. (2011). Fungal lipochitooligosaccharide symbiotic signals in arbuscular mycorrhiza. Nature. 469:

98 Πως γίνεται η απορρόφηση φωσφορικών από τα φυτά; 1. Μονοπάτι «άμεσης» πρόσληψης: Έδαφος Ριζοδερμίδα 2. Μονοπάτι της μυκόρριζας: Έδαφος Μύκητας Ρίζα Σε κάθε μονοπάτι: διαφορετικοί μεταφορείς φωσφορικών.

99 Αφομοίωση μεταφορά βαρέων μετάλλων Κινητοποίηση μετάλλων στη ριζόσφαιρα (έκκριση οργανικών οξέων /φυτοσιδηροφόρων) Μεταφορά και δημιουργία συμπλόκων στο κυτταρόπλασμα - αποτοξινωση ή/και μεταφορά με πρόσδεση σε ligands, π.χ. οργανικά οξέα, αμινοξέα, - σε πολυπεπτίδια, π.χ. HSP, - σε μη πρωτεϊνικά πεπτίδια-θειόλες φυτοχηλατίνες) Αποθήκευση στα χυμοτόπια Εκλυση στην ατμόσφαιρα-

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101 Φυτο-αποκατάσταση