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At the first lecture it was clear that many of you understood little of what I said. Two problems are : (i) (i)English (ii) New words, new concepts, that.

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Presentation on theme: "At the first lecture it was clear that many of you understood little of what I said. Two problems are : (i) (i)English (ii) New words, new concepts, that."— Presentation transcript:


2 At the first lecture it was clear that many of you understood little of what I said. Two problems are : (i) (i)English (ii) New words, new concepts, that make you stumble... But other problems could be : (i) If you are a first year student then you are not sure yet of what is expected of you as a student. (ii) You have prior expectations of what you will learn in this General Education course, and these expectations are different to what you are getting. Such an attitude will prevent you from learning.

3 There is a solution ! 1. Stop being a high school student and start being a university student. 2. Think with flexibility. 3. 3.Accept that you may not understand a concept now, but that you may later. 4. 4.If you come across a word or concept that you don’t understand, then do not stumble at that word or concept, but step over it and continue to listen. You can come back to it later. If you get stuck at one place then you cannot learn anything.

4 5. 5.READ AS MUCH AS POSSIBLE. It is your duty to start reading and to continue to read the rest of your life. 6. 6.We cannot teach you to learn, you must learn by yourself. 7. 7.The best students are those driven by CURIOSITY. 8. The best students do not expect an answer to all of their questions; and yet expect many answers to one question.

5 9. 9.Expect to be educated very widely at ICU, but also expect the opportunity to learn some things at great depth. Above all, broaden your horizons by learning about many things. A broad knowledge of many things, and a specialised knowledge of some will help you in later life. But the most important is learning how to learn. 10. 10.Expect to increase your vocabulary by 100%, that is to double your vocabulary in your four years as a student. 11. All the same, you can also expect to get a great deal of enjoyment from the university environment, and make many friends. However, never forget your duty to learn and do well.

6 Now a thought test ! I want to see how much you are aware of your surroundings, especially the biosphere, because what I will introduce next relates to the biosphere.

7 Gas (Symbol)Percentage Nitrogen (N)78.03 Oxygen (O)20.99 Argon (Ar).94 Carbon dioxide (CO2)..035 -.04 Hydrogen (H).01 Neon (Ne).012 Helium (He).0005 Krypton (Kr).0001 Ozone (O3).00006 Xenon (Xe).000009

8 3. TODAY: Biogeochemical cycling. The four important elements in the biosphere related to life. Why N is an important macronutrient. 4. Next Wednesday: The phenomenon of the Rhizobium/legume symbiosis 5. Next Friday: How plant cells work, the biology of the root hair 6. The following Monday: Experiments with root hairs 7. The following Tuesday: Agrobacterium and science


10 Biogeochemical Cycling: movement of elements within or between ecosystems caused by organisms, by geological, hydrological, and atmospheric forces, and by chemical reactions Elements within a cycle can move as: Solids, Liquids, or Gases The chemical form of an element can vary among physical states or within a physical state (e.g., Nitrogen as NH 4 +, NH 3, N 2 ) Elements involved in cycling can also have different chemical forms (e.g., Carbon as CO 2 (carbon dioxide) or CO (carbon monoxide)

11 There are two basic terms used in cycling: Pools: the amount of an element within a physical location or component of a cycle (sinks) e.g.: tree, ocean, atmosphere, soil Fluxes: the rate of movement of an element between pools e.g.: evaporation, burning, dissolution of a rock, river carrying materials from land to the ocean

12 Residence Time The length of time that an atom or molecule of a particular element spends in a particular location or component of a cycle Mean Residence Time Altering cycling rates can alter mean residence times and have the potential to lead to either depletion or pollution Recycle Time

13 Basics of nutrient cycling Short-term: fixed stock available for organisms; question of turnover time Long-term: exchange between short-term pools and minerals/fossils Anthropogenic perturbations of nutrient cycling, local and global

14 Local and global cycling Local vs. global feedbacks in nutrient dynamics (recycling vs. one-way flow) depend on physics: gaseous phases mix, solid/liquid phases stay –one-way: energy, water –partly recycled: carbon, nitrogen –mostly recycled: phosphorus Humans have long affected local landscapes, but only recently affected global biogeochemical cycles

15 Dynamics of recycled nutrients cycling: available, temporarily unavailable, incorporated amount of biomass in plants = biomass:nutrient ratio (approx. 2:1 for carbon, varies more for other nutrients) fast cycling can be good for organisms (lots of available nutrient), but can also lead to long- term nutrient losses organisms unavailable (organic) available (mineralized) death decomposition/ mineralization uptake

16 The water cycle water goes through biological systems on essentially a one-way trip cycle is fairly quick (except for aquifers, deep ocean circulation)

17 Let’s consider four biogeochemical cycles of elements required by organisms for life Carbon Phosphorus Sulphur Nitrogen

18 The Carbon Cycle Carbon Forms: CO 2, CO, CH 4, H 2 CO 3, organic matter, CaCO 3

19 Major pools of the carbon cycle in billions of tons of carbon. The oceans contain the largest pool of carbon.

20 Recycling rate of H 2 O, O 2 and CO 2 among the atmosphere, hydrosphere, biosphere and lithosphere

21 Carbon cycle Central “nutrient”: –closely bound to energy –bound to N –makes up structure of most organisms: 50% of dry biomass Major carbon storage, or sinks: – – slow-decomposing compounds in soil – – bicarbonate in the ocean – – fossil fuels – – wood

22 Carbon (2) Gaseous phase: well-mixed. Atmospheric concentration  350 ppm (pre-industrial  250 ppm) Aqueous phase: dissolves in ocean water (bicarbonate buffer). Solid phase: residence times of carbon in soil, and in plants, from weeks to centuries

23 The Phosphorus Cycle The phosphorus cycle is much slower than that of C or N

24 Phosphorus Extremely local recycling (no gaseous phase) Long-term weathering/erosion cycle Most important/limiting in aquatic ecosystems, tropical terrestrial habitats

25 The Sulphur Cycle

26 Dimethylsulfide (DMS) is released by phytoplankton, is then oxidized to sulfur dioxide and ultimately sulfate in the atmosphere. Sulfate can cause clouds to form by having water droplets condense on it.

27 The Nitrogen Cycle

28 Nitrogen Nitrogen: used for proteins, RUBISCO only nitrates (and some ammonium) available to plants: N mineralization by decomposers long-term loss/gain of N: deposition, leaching, volatilization

29 Nitrogen (2) Nitrogen dynamics depend on plant chemistry (C:N ratio, pH, decomposability), carbon dynamics, microbial community Short-cuts for plants: N-fixing organisms, mycorrhizae, direct uptake of organic N (?) Human nitrogen loading: fertilizer runoff (overflows from previously almost-closed cycles)

30 What’s so important about Nitrogen cycling? essential nutrient (fertilizers, growing legumes as crops) – changes in native species composition of ecosystem atmospheric pollutant (burning fuels) groundwater pollutant



33 Forms of Organic N Protein & peptide N Labile N Hydrolyzable Unknown N Acid Insoluble N Amino sugar N Nucleic acid N

34 Major Inorganic N Compounds CompoundFormulaOxidation state Form in soil AmmoniumNH 4 + -3Fixed in clay lattice, dissolved, as gaseous ammonia (NH 3 ) Hydroxylamine Dinitrogen Nitrous oxide Nitric oxide Nitrite Nitrate NH 2 OH N 2 N 2 O NO NO 2 - NO 3 - 0 +1 +2 +3 +5 Not detected Gas Gas, dissolved Dissolved

35 Nitrogen Fixation The nodules on the roots of this bean plant contain bacteria called Rhizobium that help convert nitrogen in the soil to a form the plant can utilize.

36 Dinitrogen Fixation TreatmentYield (g) OatsPeas No N added Non-inoculated Inoculated with legume soil Inoculated with sterile soil 0.6 0.7 — 0.8 16.4 0.9 112 mg NO 3 - –N per pot added Non-inoculated Inoculated with legume soil 12.0 11.6 12.9 15.3 Hellriegel and Wilfarth (1888) The alder, whose fat shadow nourisheth– Each plant set neere him long flourisheth. –William Browne (1613), Brittania’s Pastorals, Book I, Song 2

37 Types of Biological Nitrogen Fixation Free-living (asymbiotic) Cyanobacteria Azotobacter Associative Rhizosphere–Azospirillum Lichens–cyanobacteria Leaf nodules Symbiotic Legume-rhizobia Actinorhizal-Frankia

38 Free-living N 2 Fixation Energy 20-120 g C used to fix 1 g N Combined Nitrogen nif genes tightly regulated Inhibited at low NH 4 + and NO 3 - (1 μg g -1 soil, 300 μM) Oxygen Avoidance (anaerobes) Microaerophilly Respiratory protection Specialized cells (heterocysts, vesicles) Spatial/temporal separation Conformational protection

39 Associative N 2 Fixation Phyllosphere or rhizosphere (tropical grasses) Azosprillum, Acetobacter 1 to 10% of rhizosphere population Some establish within root Same energy and oxygen limitations as free-living Acetobacter diazotrophicus lives in internal tissue of sugar cane, grows in 30% sucrose, can reach populations of 10 6 to 10 7 cells g -1 tissue, and fix 100 to 150 kg N ha -1 y -1

40 Estimated Average Rates of Biological N 2 Fixation Organism or systemN 2 fixed (kg ha -1 y -1 ) Free-living microorganisms Cyanobacteria Azotobacter Clostridium pasteurianum 25 0.3 0.1-0.5 Grass-Bacteria associative symbioses Azospirillum5-25 Cyanobacterial associations Gunnera Azolla Lichens 10-20 300 40-80 Leguminous plant symbioses with rhizobia Grain legumes (Glycine, Vigna, Lespedeza, Phaseolus) Pasture legumes (Trifolium, Medicago, Lupinus) 50-100 100-600 Actinorhizal plant symbioses with Frankia Alnus Hippophaë Ceanothus Coriaria Casuarina 40-300 1-150 1-50 50-150 50

41 Nitrogen Fixation Almost all N is in the atmosphere 90-190 Tg N fixed by terrestrial systems 40-200 Tg N fixed by aquatic systems 3-10 Tg N fixed by lightning 32-53 Tg N fixed by crops

42 Some biogeochemical cycling key points: cycling occurs at local to global scales biogeochemical cycles have 2 basic parts: pools and fluxes elements are recycled among the biosphere, atmosphere, lithosphere and hydrosphere cycles of each element differ (chemistry, rates, pools, fluxes, interactions) cycling is important because it can affect many other aspects of the environment and the quality of our lives

43 Nitrogen Metabolism

44 Plant Nutrition Plant metabolism is based on sunlight and inorganic elements present in water, air, and soil. C, H, and O and energy are used to generate organic molecules via photosynthesis. Other chemical elements, such as mineral nutrients, are also absorbed from soil.

45 Plant Nutrients Plants absorb many elements, some of which they do not need. An element is considered an essential nutrient if it meets three criteria: It is necessary for complete, normal plant development through a full life cycle. It itself is necessary; no substitute can be effective. It must be acting within the plant, not outside it. Many roles in plant metabolism.

46 Types of Essential Nutrients Nine essential nutrients, called macronutrients, are needed in very large amounts Eight other essential nutrients, called micronutrients, are needed only in small amounts.

47 Essential Nutrients to Most Plants Macronutrient % Dry Weight Component/Function Carbon (C )45.0 Organic compounds Oxygen (O)45.0 Organic compounds Hydrogen (H)6.0 Organic compounds Nitrogen (N)1.0-4.0 Amino acids; nucleic acids, chlorophyll Potassium (K)1.0 Amino acids; regulates stomata opening/closing Calcium (Ca)0.5 Enzyme cofactor; influences cell permeability Phosphorus (P)0.2 ATP; proteins; nucleic acids; phosphoplipids Magnesium (Mg)0.2 Chlorophyll; enzyme activator Sulfur (S)0.1 CoA; amino acids

48 Essential Nutrients to Most Plants MicronutrientComponent/Function Iron (Fe) Cytochromes; chlorophyll synthesis Chlorine (Cl) Osmosis; water-splitting in photosynthesis Copper (Cu) Plastocyanin; enzyme activator Manganese (Mn) Enzyme activator; component of chlorophyll Zinc (Zn) Enzyme activator Molybdenum (Mo) Nitrogen fixation Boron (B) Cofactor in chlorophyll synthesis Nickel (Ni) Cofactor for enzyme functioning in nitrogen metabolism

49 Nitrogen: An Essential Macronutrient N is not present in rock, but is abundant in the atmosphere as a gas, N 2. The process of converting N 2 to chemically active forms of N is nitrogen metabolism. Nitrogen metabolism consists of 3 stages: Nitrogen Fixation (N 2 -> NO 3 - ) Nitrogen Reduction (NO 3 - -> NO 2 - - > NH 3 - > NH 4 + ) Nitrogen Assimilation (transfer of NH 2 groups) Runoff, leaching, denitrification, and harvested crops reduce soil nitrogen.

50 Nitrogen Cycling Processes Nitrogen Fixation – bacteria convert nitrogen gas (N 2 ) to ammonia (NH 3 ). Decomposition – dead nitrogen fixers release N- containing compounds. Ammonification – bacteria and fungi decompose dead plants and animals and release excess NH 3 and ammonium ions (NH 4 + ). Nitrification – type of chemosynthesis where NH 3 or NH 4 + is converted to nitrite (NO 2 -) ; other bacteria convert NO 2 - to nitrate (NO 3 -). Denitrification – bacteria convert NO 2 - and NO 3 - to N 2.

51 Means of Nitrogen Fixation 1) Human manufacturing of synthetic fertilizers 2) Lightning 3) Nitrogen-fixing bacteria and cyanobacteria

52 Some are free-living in soil (E.g., Nostoc, Azotobacter); others live symbiotically with plants (E.g., Frankia, Rhizobium). These organisms have nitrogenase, an enzyme that uses N 2 as a substrate. N 2 + 8e - + 8H + + 16ATP -> 2NH 3 + H 2 + 16ADP + 16P i NH 3 is immediately converted to NH 4+. Bacterial enzymes sensitive to O 2. Leghemoglobin binds to O 2 and protects enzymes. Symbiotic fixation rate depends on plant stage. Nitrogen Fixing Bacteria and Cyanobacteria

53 Natural Sources of Organic N Source% N Dried blood12 Peruvian guano12 Dried fish meal10 Peanut meal7 Cottonseed meal7 Sludge from sewer treatment plant6 Poultry manure5 Bone meal4 Cattle manure2

54 Symbiotic Nitrogen Fixation Nitrogen-fixing bacteria fix N (E.g., Rhizobium) Plants fix sugars (E.g.,legumes). Plants form swellings that house N-fixing bacteria, called root nodules. Mutualistic association. Excess NH 3 is released into soil. Crop rotation maintains soil fertility.

55 Development of a Root Nodule Bacteria enter the root through an infection thread. Bacteria are then released into cell and assume form called bacteroids, contained within vesicles.

56 Symbiotic Nitrogen Fixation The Rhizobium-legume association Bacterial associations with certain plant families, primarily legume species, make the largest single contribution to biological nitrogen fixation in the biosphere


58 When this association is not present or functional, we apply nitrogen-containing fertilizers to replace reduced nitrogen removed from the soil during repeated cycles of crop production. This practice consumes fossil fuels, both in fertilizer production and application.

59 Biological nitrogen fixation is the reduction of atmospheric nitrogen gas (N 2 ) to ammonium ions (NH 4 + ) by the oxygen-sensitive enzyme, nitrogenase. Reducing power is provided by NAPH/ferredoxin, via an Fe/Mo centre. Even within the bacteria, only certain free-living bacteria (Klebsiella, Azospirillum, Azotobacter), blue-green bacteria (Anabaena) and a few symbiotic Rhizobial species are known nitrogen-fixers. Plant genomes lack any genes encoding this enzyme, which occurs only in prokaryotes (bacteria). Another nitrogen-fixing association exists between an Actinomycete (Frankia spp.) and alder (Alnus spp.)

60 The enzyme nitrogenase catalyses the conversion of atmospheric, gaseous dinitrogen (N 2 ) and dihydrogen (H 2 ) to ammonia (NH 3 ), as shown in the chemical equation below: N 2 + 3 H 2  2 NH 3 The above reaction seems simple enough and the atmosphere is 78% N 2, so why is this enzyme so important? The incredibly strong (triple) bond in N 2 makes this reaction very difficult to carry out efficiently. In fact, nitrogenase consumes ~16 moles of ATP for every molecule of N 2 it reduces to NH 3, which makes it one of the most energy-expensive processes known in Nature.

61 Fe - S - Mo electron transfer cofactor in nitrogenase S Fe Mo homocitrate


63 Biological NH 3 creation (nitrogen fixation) accounts for an estimated 170 x 10 9 kg of ammonia every year. Human industrial production amounts to some 80 x 10 9 kg of ammonia yearly. The industrial process (Haber-Bosh process) uses an Fe catalyst to dissociate molecules of N 2 to atomic nitrogen on the catalyst surface, followed by reaction with H 2 to form ammonia. This reaction typically runs at ~450º C and 500 atmospheres pressure. These extreme reaction conditions consume a huge amount of energy each year, considering the scale at which NH 3 is produced industrially.

64 If a way could be found to mimic nitrogenase catalysis (a reaction conducted at 0.78 atmospheres N 2 pressure and ambient temperatures), huge amounts of energy (and money) could be saved in industrial ammonia production. If a way could be found to transfer the capacity to form N-fixing symbioses from a typical legume host to an important non-host crop species such as corn or wheat, far less fertilizer would be needed to be produced and applied in order to sustain crop yields The Dream…..

65 Because of its current and potential economic importance, the interaction between Rhizobia and leguminous plants has been intensively studied. Our understanding of the process by which these two symbionts establish a functional association is still not complete, but it has provided a paradigm for many aspects of cell-to-cell communication between microbes and plants (e.g. during pathogen attack), and even between cells within plants (e.g. developmental signals; fertilization by pollen).

66 Symbiotic Rhizobia are classified in two groups: Fast-growing Rhizobium spp. whose nodulation functions (nif, fix) are encoded on their symbiotic megaplasmids (pSym) Slow-growing Bradyrhizobium spp. whose N-fixation and nodulation functions are encoded on their chromosome. There are also two types of nodule that can be formed: determinate and indeterminate This outcome is controlled by the plant host

67 Formed on tropical legumes by Rhizobium and Bradyrhizobium Meristematic activity not persistent - present only during early stage of nodule formation; after that, cells simply expand rather than divide, to form globose nodules. Nodules arise just below epidermis; largely internal vascular system Determinate nodules

68 Uninfected cells dispersed throughout nodule; equipped to assimilate NH 4 + as ureides (allantoin and allantoic acid) allantoinallantoic acid

69 Indeterminate nodules Formed on temperate legumes (pea, clover, alfalfa); typically by Rhizobium spp. Cylindrical nodules with a persistent meristem; nodule growth creates zones of different developmental stages Nodule arises near endodermis, and nodule vasculature clearly connected with root vascular system

70 Uninfected cells of indeterminate nodules assimilate NH 4 + as amides (asparagine, glutamine)

71 Typical Associations (cross-inoculation groups) Rhizobium leguminosarum biovar phaseoli colonizes bean (Phaseolus spp.) (tropical; determinate nodules) R.l. biovar trifolii colonizes clover (Trifolium spp.) (temperate; indeterminate nodules) R.l. biovar viciae colonizes pea (Pisum spp.) and vetch (temperate; indeterminate nodules)

72 Rhizobium meliloti colonizes alfalfa (Medicago sativa) temperate; indeterminate nodules Rhizobium fredii colonizes soybean (Glycine max) tropical; determinate nodules Bradyrhizobium japonicum colonizes soybean tropical; determinate nodules Rhizobium NGR 234 colonizes Parasponia and tropicals; very broad host range

73 Nodule development process 1. Bacteria encounter root; they are chemotactically attracted toward specific plant chemicals (flavonoids) exuding from root tissue, especially in response to nitrogen limitation daidzein (an isoflavone) naringenin (a flavanone)

74 2. Bacteria attracted to the root attach themselves to the root hair surface and secrete specific oligosaccharide signal molecules (nod factors). nod factor

75 Examples of different nod factors

76 3. In response to oligosaccharide signals, the root hair becomes deformed and curls at the tip; bacteria become enclosed in small pocket. Cortical cell division is induced within the root.


78 4. Bacteria then invade the root hair cell and move along an internal, plant-derived “infection thread”, multiplying, and secreting polysaccharides that fill the channel.

79 Rhizobium cells expressing GFP (green fluorescent protein) invade a host root hair infection thread

80 5. Infection thread penetrates through several layers of cortical cells and then ramifies within the cortex. Cells in advance of the thread divide and organize themselves into a nodule primordium. 6. The branched infection thread enters the nodule primordium zone and penetrates individual primordium cells. 7. Bacteria are released from the infection thread into the cytoplasm of the host cells, but remain surrounded by the peribacteroid membrane. Failure to form the PBM results in the activation of host defenses and/or the formation of ineffective nodules.

81 8. Infected root cells swell and cease dividing. Bacteria within the swollen cells change form to become endosymbiotic bacteroids, which begin to fix nitrogen. The nodule provides an oxygen-controlled environment (leghemoglobin = pink nodule interior) structured to facilitate transport of reduced nitrogen metabolites from the bacteroids to the plant vascular system, and of photosynthate from the host plant to the bacteroids.

82 Sinorhizobium meliloti Bacteroids

83 peribacteroid membrane bacteroid transporters

84 Types of bacterial functions involved in nodulation and nitrogen fixation nod (nodulation) and nol (nod locus) genes mutations in these genes block nodule formation or alter host range most have been identified by transposon mutagenesis, DNA sequencing and protein analysis, in R. meliloti, R. leguminosarum bv viciae and trifolii fall into four classes: nodD nodA, B and C (common nod genes) hsn (host-specific nod genes) other nod genes

85 F G H I N D 1 A B C I J Q P G E F H D 3 E K D H A B C (nol)(nod)(nif) (fix) Gene clusters on R. meliloti pSym plasmid N M L R E F D A B C I J T C B A H D K E N Gene clusters on R. leguminosarum bv trifolii pSym plasmid - - - D 2 D 1 Y A B C S U I J - - - Gene cluster on Bradyrhizobium japonicum chromosome

86 Nod D (the sensor) the nod D gene product recognizes molecules (phenylpropanoid-derived flavonoids) produced by plant roots and becomes activated as a result of that binding activated nodD protein positively controls the expression of the other genes in the nod gene “regulon” (signal transduction) different nodD alleles recognize various flavonoid structures with different affinities, and respond with differential patterns of nod gene activation naringenin (a flavanone)

87 Common nod genes - nod ABC mutations in nodA,B or C completely abolish the ability of the bacteria to nodulate the host plant; they are found as part of the nod gene “regulon” in all Rhizobia (  common) products of these genes are required for bacterial induction of root cell hair deformation and root cortical cell division

88 The nod ABC gene products are enzymes responsible for synthesis of diffusible nod factors, whcih are sulfated and acylated beta-1,4-oligosaccharides of glucosamine (other gene products, e.g. NodH, may also be needed for special modifications) [ 1, 2 or 3 ] [C16 or C18 fatty acid] SO 3 =

89 nod factors are active on host plants at very low concentrations (10 -8 to 10 -11 M) but have no effect on non-host species

90 mutations in these genes elicit abnormal root reactions on their usual hosts, and sometimes elicit root hair deformation reactions on plants that are not usually hosts Host-specific nod genes Example: loss of nodH function in R. meliloti results in synthesis of a nod factor that is no longer effective on alfalfa but has gained activity on vetch The  nodH nod factor is now more hydrophobic than the normal factor - no sulfate group on the oligosaccharide. The role of the nodH gene product is therefore to add a specific sulfate group, and thereby change host specificity

91 Other nod genes May be involved in the attachment of the bacteria to the plant surface, or in export of signal molecules, or proteins needed for a successful symbiotic relationship

92 exo (exopolysaccharide) genes In Rhizobium-legume interactions that lead to indeterminate nodules, exo mutants cannot invade the plant properly. However, they do provoke the typical plant cell division pattern and root deformation, and can even lead to nodule formation, although these are often empty (no bacteroids). In interactions that usually produce determinate nodules, exo mutations tend to have no effect on the process. Exopolysaccharides may provide substrate for signal production, osmotic matrix needed during invasion, and/or a recognition or masking function during invasion Encode proteins needed for exopolysaccharide synthesis and secretion

93 example of Rhizobial exopolysaccharide


95 nif (nitrogen fixation) genes Gene products are required for symbiotic nitrogen fixation, and for nitrogen fixation in free-living N-fixing species Example: subunits of nitrogenase


97 fix (fixation) genes Gene products required to successfully establish a functional N-fixing nodule. No fix homologues have been identified in free-living N-fixing bacteria. Example: regulatory proteins that monitor and control oxygen levels within the bacteroids

98 FixL senses the oxygen level; at low oxygen tensions, it acts as a kinase on FixJ, which regulates expression of two more transcriptional regulators: NifA, the upstream activator of nif and some fix genes; FixK, the regulator of fixN (another oxgen sensor?) This key transducing protein, FixL, is a novel hemoprotein kinase with a complex structure. It has an N-terminal membrane-anchoring domain, followed by the heme binding section, and a C-terminal kinase catalytic domain. Result? Low oxygen tension activates nif gene transcription and permits the oxygen-sensitive nitrogenase to function.

99 Metabolic genes and transporters Dicarboxylic acid (malate) transport and metabolism Genes for other functions yet to be identified….  Proteomic analysis of bacteroids and peribacteroid membrane preparations  DNA microarray analysis of gene expression patterns


101 Host plant role in nodulation 1. Production and release of nod gene inducers - flavonoids 2. Activation of plant genes specifically required for successful nodule formation - nodulins 3. Suppression of genes normally involved in repelling microbial invaders - host defense genes

102 Nodulins Bacterial attachment Root hair invasion Bacteroid development Nitrogen fixation Nodule senescence early nodulins late nodulins Nodulins? Pre-infection Infection and nodule formation Nodule function and maintenance Nodule senescence

103 Early nodulins At least 20 nodule-specific or nodule-enhanced genes are expressed in plant roots during nodule formation; most of these appear after the initiation of the visible nodule. Five different nodulins are expressed only in cells containing growing infection threads. These may encode proteins that are part of the plasmalemma surrounding the infection thread, or enzymes needed to make or modify other molecules

104 Twelve nodulins are expressed in root hairs and in cortical cells that contain growing infection threads. They are also expressed in host cells a few layers ahead of the growing infection thread. Late nodulins The best studied and most abundant late nodulin is the protein component of leghemoglobin. The heme component of leghemoglobin appears to be synthesized by the bacteroids.

105 Treatment of Lotus japonicus roots with nod factor from Mesorhizobium loti (NF), or infection with wt M. loti, (+) or an ineffective  nodC strain of M. loti (-)

106 Other late nodulins are enzymes or subunits of enzymes that function in nitrogen metabolism (glutamine synthetase; uricase) or carbon metabolism (sucrose synthase). Others are associated with the peribacteroid membrane, and probably are involved in transport functions. These late nodulin gene products are usually not unique to nodule function, but are found in other parts of the plant as well. This is consistent with the hypothesis that nodule formation evolved as a specialized form of root differentiation.

107 There must be many other host gene functions that are needed for successful nodule formation. Example: what is the receptor for the nod factor? These are being sought through genomic and proteomic analyses, and through generation of plant mutants that fail to nodulate properly The full genome sequencing of Medicago truncatula and Lotus japonicus, both currently underway, will greatly speed up this discovery process.

108 A plant receptor-like kinase required for both bacterial and fungal symbiosis S. Stracke et al Nature 417:959 (2002) Screened mutagenized populations of the legume Lotus japonicus for mutants that showed an inability to be colonized by VAM Mutants found to also be affected in their ability to be colonized by nitrogen-fixing bacteria (“symbiotic mutants”)

109 WTmutant

110 Mutant


112 LRRs PM Protein kinase catalytic domain cytosol






118 Inhibitor of nodulation Inducers of nodulation in Rhizobium leguminosarum bv viciae luteolin eriodictyol genistein Figure 19.70


120 Genetics of Nitrogenase GeneProperties and function nifH nifDK nifA nifB nifEN nifS fixABCX fixK fixLJ fixNOQP fixGHIS Dinitrogenase reductase Dinitrogenase Regulatory, activator of most nif and fix genes FeMo cofactor biosynthesis Unknown Electron transfer Regulatory Regulatory, two-component sensor/effector Electron transfer Transmembrane complex

121 Nitrogenase FeMo Cofactor N 2 + 8H + 2NH 3 + H 2 8e-8e- 4C 2 H 2 + 8H + 4C 2 H 2 Dinitrogenase reductase Fd(red) Fd(ox) nMgATP nMgADP + nP i N 2 + 8H + + 8e - + 16 MgATP  2NH 3 + H 2 + 16MgADP

122 Taxonomy of Rhizobia GenusSpeciesHost plant Rhizobiumleguminosarum bv. trifolii “ bv. viciae “ bv. phaseoli tropici etli Trifolium (clovers) Pisum (peas), Vicia (field beans), Lens (lentils), Lathyrus Phaseolus (bean) Phaseolus (bean), Leucaena Phaseolus (bean) Sinorhizobiummeliloti fredii saheli teranga Melilotus (sweetclover), Medicago (alfalfa), Trigonella Glycine (soybean) Sesbania Sesbania, Acacia Bradyrhizobiumjaponicum elkanii liaoningense Glycine (soybean) AzorhizobiumcaulinodansSesbania (stem nodule) ‘Meso rhizobium’loti huakuii ciceri tianshanense mediterraneum Lotus (trefoil) Astragalus (milkvetch) Cicer (chickpea) [Rhizobium]galegaeGalega (goat’s rue), Leucaena Photorhizobiumspp.Aeschynomene (stem nodule)

123 Nitrogen Fixation Energy intensive process : N 2 + 8H+ + 8e - + 16 ATP = 2NH 3 + H 2 + 16ADP + 16 Pi Performed only by selected bacteria and actinomycetes Performed in nitrogen fixing crops (ex: soybeans)

124 Nodulation in Legumes

125 Role of Root Exudates General Amino sugars, sugars Specific Flavones (luteolin), isoflavones (genistein), flavanones, chalcones Inducers/repressors of nod genes Vary by plant species Responsiveness varies by rhizobia species

126 nod Gene Expression Common nod genes Nod factor–LCO (lipo-chitin oligosaccharide)

127 Infection Process Attachment Root hair curling Localized cell wall degradation Infection thread Cortical cell differentiation Rhizobia released into cytoplasm Bacterioid differentiation (symbiosome formation) Induction of nodulins

128 Nodule Metabolism Oxygen metabolism Variable diffusion barrier Leghemoglobin Nitrogen metabolism NH 3 diffuses to cytosol Assimilation by GOGAT Conversion to organic-N for transport Carbon metabolism Sucrose converted to dicarboxylic acids Functioning TCA in bacteroids C stored in nodules as starch

129 Nitrogen Cycle

130 Sources Lightning Inorganic fertilizers Nitrogen Fixation Animal Residues Crop residues Organic fertilizers


132 Forms of Nitrogen Urea  CO(NH 2 ) 2 Ammonia  NH 3 (gaseous) Ammonium  NH 4 Nitrate  NO 3 Nitrite  NO 2 Atmospheric Dinitrogen  N 2 Organic N

133 Global Nitrogen Reservoirs Nitrogen Reservoir Metric tons nitrogen Actively cycled Atmosphere3.9*10 15 No Ocean  soluble salts Biomass 6.9*10 11 5.2*10 8 Yes Land  organic matter  Biota 1.1*10 11 2.5*10 10 Slow Yes

134 Roles of Nitrogen Plants and bacteria use nitrogen in the form of NH 4 + or NO 3 - It serves as an electron acceptor in anaerobic environment Nitrogen is often the most limiting nutrient in soil and water.

135 Nitrogen is a key element for amino acids nucleic acids (purine, pyrimidine) cell wall components of bacteria (NAM).

136 Nitrogen Cycles Ammonification/mineralization Immobilization Nitrogen Fixation Nitrification Denitrification

137 Mineralization or Ammonification Decomposers: earthworms, termites, slugs, snails, bacteria, and fungi Uses extracellular enzymes  initiate degradation of plant polymers Microorganisms uses: Proteases, lysozymes, nucleases to degrade nitrogen containing molecules

138 Plants die or bacterial cells lyse  release of organic nitrogen Organic nitrogen is converted to inorganic nitrogen (NH 3 ) When pH<7.5, converted rapidly to NH 4 Example: Urea NH 3 + 2 CO 2

139 Immobilization The opposite of mineralization Happens when nitrogen is limiting in the environment Nitrogen limitation is governed by C/N ratio C/N typical for soil microbial biomass is 20 C/N < 20  Mineralization C/N > 20  Immobilization

140 Microorganisms fixing Azobacter Beijerinckia Azospirillum Clostridium Cyanobacteria Require the enzyme nitrogenase Inhibited by oxygen Inhibited by ammonia (end product)

141 Rates of Nitrogen Fixation N 2 fixing systemNitrogen Fixation (kg N/hect/year) Rhizobium-legume200-300 Cyanobacteria- moss30-40 Rhizosphere associations 2-25 Free- living1-2

142 Bacterial Fixation Occurs mostly in salt marshes Is absent from low pH peat of northern bogs Cyanobacteria found in waterlogged soils

143 Denitrification Removes a limiting nutrient from the environment 4NO 3 - + C 6 H 12 O 6  2N 2 + 6 H 2 0 Inhibited by O 2 Not inhibited by ammonia Microbial reaction Nitrate is the terminal electron acceptor

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