1. Nitrogen assimilation Plant Physiol Biotech, Biol 3470 Feb. 28, 2006 Lecture 12 Chapter 8

2. Nitrogen: an essential element Fourth most common element –Proteins, NAs, PGRs, chlorophyll,… Bioavailable forms: nitrate (NO 3 - ) and ammonia (NH 4 + ) Paradox: limiting in environment for growth but plenty available in atmosphere as N 2 –Biologically unavailable! –Need prokaryotes to help with this… wps.prenhall.com

3. The N cycle circulates N in the biosphere 3 major N pools: –atmosphere, soil, biomass plants convert inorganic soil N to organic N (amino acids, NAs, etc.) Organic N moves up the chain to animals (they eat plants!) Returns to soil in animal waste and decomposition after death Fig 8.1 inorganic Organic N pools Nitrification by bacteria by prokaryotes and fungi or mineralization

4. Essential N cycle processes include ammonification, nitrification and denitrification Ammonification by prokaryotes and fungi returns N to soil: organic N  ammonia (NH 4 + ) Make ammonia biologically available by its sequential oxidization to nitrite and nitrate by soil bacteria during nitrification Plants must compete for nitrate with soil bacteria that reduce NO 3 - to N 2 : denitrification –93 to 130 Mt / year back to atmosphere

5. N fixation reduces N 2 to NH 4 + Soil N pool loses N to atmosphere but regains N through action of fixing bacteria N 2  NH 4 + does not happen spontaneously: highly endergonic = very energetically costly Where does the biologically available soil N come from? 10% of N fixed into N oxides: lightning, UV, air pollution 30% via industrial N fixation: make N using fossil fuels via Haver-Bosch process at high T and pressure 60% via biological N fixation by microorganisms: poorly understood but extremely valuable process

6. Only prokaryotes are nitrogen-fixers Symbiotic relationship involves metabolic integration between specific bacterial (microsymbiont) and plant (host) species This usually results in forming nodules on the roots/stem This is seen between Rhizobia (bacteria) and legumes (plant) Need dinitrogenase (a/k/a nitrogenase) to fix/reduce N 2 directly to ammonia Can be free living or symbionts, photosynthetic or heterotrophic bacteria or cyanobacteria All need low/zero O 2 and high C levels: high energy requirement for N fixation slows growth Fig. 8.2

7. Only a small number of economically important plants can fix their own organic N These are legumes –“Pea family” (Fabaceae): lupin, clover, alfalfa, field beans and peas Note that the top 5 crops cannot fix N and are reliant on fertilizer for high yields This ability evolved over a long period of time and involves both plant and bacterial genes Engineering this trait into modern crops is thus very difficult but economically desirable Rhizobia exist as biovars that restrict the legume species with whom they can establish symbiotic relationships www.botany.hawaii.edu www.agry.purdue.edu Alfalfa (Medicago sativa)

8. Bacteria continuously multiply during infection Infection complete when bacteria released into host cells by budding off plasma membrane of infection thread Nodule keeps growing via nodule meristem (rapidly dividing cells) Bacteria multiply and infect new plant cells Establish vascular connections with plant: photoassimilate (C) in, fixed N (ammonia, AAs) out When they start fixing N 2 for the plant they are called bacteroids Rhizobia multiply and infect multiple plant cells within the developing nodule Fig. 8.3 Fig. 8.5 Infection thread

9. Oxygen inhibits dinitrogenase Irreversibly denatures both constituent proteins But need cellular respiration to make ATP! Strategies Free living bacteria maintain an anaerobic lifestyle or only fix N2 when under anaerobisis Cyanobacteria structurally isolate nitrogen fixing cells (heterocysts): thick walls, high respiratory capacity limits O2 levels, lack PSII and thus can’t evolve O2 Nodules restrict O2 to an O2-binding protein, leghemoglobin –Synthesized by host, present in bacteroid infected host cells –Keeps respiration high while sequestering O2 from dinitrogenase Fig. 8.8

10. Key metabolic step is conversion of fixed ammonia to organic N N assimilation is energetically expensive: 2 to 15% of plant’s energy production Let’s examine assimilation of N from these two molecules Fig. 8.7 Most plants can assimilate either NO 3 - or ammonia Recall that nitrifying bacteria scavenge and convert ammonia to NO 3 - –too bad, NH 4 + is the preferred form (already reduced for incorporation into organic molecules) N assimilation is reliant on a steady supply of C !!

11. Nitrogen assimilation is a series of reactions that coordinate C and N metabolism N2N2 NO 3 - Biologically unavailable! (via nitrification by bacteria or fertilizer) NH 4 + Biologically available but toxic! ATP ADP + Pi NADPH NADP + H+ dinitrogenase Nitrate reductase + nitrite reductase Glutamate α-ketoglutarate (C skeleton from ______ ) Glutamate Glutamate synthase cycle For export to N sinks ATP + NADH ADP + Pi + NAD + inhibits N2ase Uncouples ATP synthesis from e- transport Thus, plants use the Glu synthase cycle to rapidly assimilate N into organic molecules

12. Where does the fixed N go? Primarily exported via xylem: monitor by radiolabeling and examining xylem exudate Temperate legumes export asparagine Asparagine is 2 steps away from Glu and Gln siphoned from glutamate synthase cycle Making N into an exportable form consumes ~20% of C allocated to N fixation Want to export organic N with as little C attached as possible (low C:N ratio) Asparagine: 2 Glu + OAA  αKG + Asp Exported from Glu synthase cycle From PEPC Gln + Asp  Glu + Asn ATP ADP Siphoned from Glu synthase cycle export

13. NO3- is assimilated by nitrate/nitrite reductase Uptake of nitrate is an energy-dependent process involving a specific transporter protein (like for most inorganic elements!) Some of this carrier is constitutive but most is inducible upon exposure to NO 3 - (inhibited by exposure to protein synthesis inhibitors) Can store NO 3 - in vacuole, assimilate directly in roots, or translocate in xylem to leaves (sinks) for assimilation there Nitrate reductase activity coordinates N and C assimilation Its complex regulation includes mechanisms of: –light and substrate which implies a requirement for photosynthetic energy (e.g., reducing power to supply e-) –phytochrome –reversible phosphorylation by a protein kinase/phosphatase

14. N uptake rate varies with plant age Highest during early rapid growth phase N uptake declines as plant enters reproductive phase Assimilated N directed towards young, developing leaves Leaves reach their maximum N content just before full maturity Then leaves become net N exporters even though they continue to import N –a/k/a N cycling Developing seeds are strong N sinks: requirement cannot be met by soil uptake alone Steal (reallocate) N from mature leaves Fig. 8.12

15. Metabolic consequences of N cycling Most soluble leaf N is tied up in one protein: __________ ! This protein is thus an N storage protein Plants mobilize N to storage in seeds, which may reduce photosynthetic capacity In legumes, a lowered C assimilation rate reduces the capacity for N assimilation –Major limiting factor for seed yield in legumes Perennials (e.g., trees) mobilize leaf N (rubisco, chlorophyll) in the autumn and store it in the roots as storage protein –N is too precious to discard with leaves!

16. Agricultural productivity is directly dependent on bioavailable N … which depends on soil pH, temp., O 2, H 2 O Influence activity of microorganisms responsible for N assimilation N is removed with the crop each year! Farmers want to maximize productivity: most crops linearly increase yield with N applied until the critical concentration is reached Fig. 8.13

17. N fertilizers are costly Energetically: 1.5 kg oil per kg fixed N –1/3 of energy cost of a crop of maize is N fertilizer Financially for farmers Without added N, yield on a plot eventually declines to a stable, base level Natural ecosystems are also N limited 2/3 contribution from N fixers, 1/3 from atmosphere (deposition of NxOs) Most N retained in forest canopy or degraded from litter and –Leached into the soil, or –Degraded by bacteria, fungi etc. Finally convert organic N to inorganic N (NO 3 -, NH 4 + ) via mineralization (e.g., ammonification) Accompanied by immobilization: retention and use of N by decomposing organisms Net mineralization (mineralized N minus immobilized N) is available to plants

18. Rate of natural nitrification varies with environmental conditions Nitrification by bacteria = rate of adding N to soil bioavailable pool (as NO 3 -, NH 4 + ) Varies with temperature, pH, moisture, oxygen Needs a lot of O 2 because this process is energy dependent Nitrification is likely a significant source of bioavailable N; difficult to show because plants keep soil N levels low! Stored N in perennials helps plants overcome low soil NO 3 - levels