Nitrogen assimilation

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

2. Nitrogen: an essential element wps.prenhall.com • Fourth most common element • Proteins, NAs, PGRs, chlorophyll,… • Bioavailable forms: nitrate (NO3-) and ammonia (NH4+) • Paradox:limiting in environment for growth but plenty available in atmosphere as N2 • Biologically unavailable! • Need prokaryotes to help with this…

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 Nitrification by bacteria inorganic or mineralization by prokaryotes and fungi Organic N pools Fig 8.1

4. Essential N cycle processes include ammonification, nitrification and denitrification • Ammonification by prokaryotes and fungi returns N to soil: organic N  ammonia (NH4+) • 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 NO3- to N2: denitrification • 93 to 130 Mt / year back to atmosphere

5. N fixation reduces N2 to NH4+ • Soil N pool loses N to atmosphere but regains N through action of fixing bacteria • N2 NH4+ 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 • Need dinitrogenase (a/k/a nitrogenase) to fix/reduce N2 directly to ammonia • Can be free living or symbionts, photosynthetic or heterotrophic bacteria or cyanobacteria • All need low/zero O2andhigh C levels: high energy requirement for N fixation slows growth • 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) Fig. 8.2

7. Only a small number of economically important plants can fix their own organic N Alfalfa (Medicago sativa) • 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.agry.purdue.edu www.botany.hawaii.edu

8. Rhizobia multiply and infect multiple plant cells within the developing nodule • 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 N2 for the plant they are called bacteroids Infection thread Fig. 8.3 Fig. 8.5

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 Fig. 8.8 • 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

10. Key metabolic step is conversion of fixed ammonia to organic N • Most plants can assimilate either NO3- or ammonia • Recall that nitrifying bacteria scavenge and convert ammonia to NO3- • too bad, NH4+ is the preferred form (already reduced for incorporation into organic molecules) • N assimilation is energetically expensive: 2 to 15% of plant’s energy production • Let’s examine assimilation of N from these two molecules N assimilation is reliant on a steady supply of C !! Fig. 8.7

11. Nitrogen assimilation is a series of reactions that coordinate C and N metabolism N2 Biologically unavailable! NO3- (via nitrification by bacteria or fertilizer) ATP NADPH dinitrogenase Nitrate reductase + nitrite reductase ADP + Pi NH4+ NADP + H+ Biologically available but toxic! α-ketoglutarate (C skeleton from ______ ) • inhibits N2ase • Uncouples ATP synthesis from e- transport • Thus, plants use the Glu synthase cycle to rapidly assimilate N into organic molecules Glutamate synthase cycle ATP + NADH ADP + Pi + NAD+ Glutamate Glutamate For export to N sinks

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 ADP ATP Glu + OAA  αKG + Asp Gln + Asp  Glu + Asn Exported from Glu synthase cycle From PEPC Siphoned from Glu synthase cycle export • 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

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 inducibleupon exposure to NO3- (inhibited by exposure to protein synthesis inhibitors) • Can store NO3- 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., O2, H2O • 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 (NO3-, NH4+) 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 NO3-, NH4+) • Varies with temperature, pH, moisture, oxygen • Needs a lot of O2 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 NO3- levels