Nitrogen is essential for plant growth but is often limited in availability. Plants have evolved symbiotic relationships with nitrogen-fixing bacteria to access atmospheric nitrogen. These relationships include root and stem nodule symbioses with rhizobia and Frankia bacteria that live inside root nodules. Cyanobacteria also engage in nitrogen-fixing symbioses, living inside specialized plant tissues. Associative bacteria in the rhizosphere can also increase nitrogen availability through nitrogen fixation. These symbioses allowed plants to colonize land and overcome nitrogen limitations.

1. Importance of Nitrogen to plants: Key Concepts
Elements essential for life: C, H, O, N, S, P, and others in smaller
quantities: K, Na, Mg, Mn, Fe, Mo, Cl, etc.
N up to 2% of dried plant biomass
Atmospheric N2 not available to organisms. Why?
Lack of N availability a major limiting factor of plant growth
400Ma plants invaded land, likely with fungal (mycorhizal) symbionts
Define:
Biogeochemistry
Immobilization
Mineralization
Diazotroph

2. Atmospheric
N2
Nitrate
NO3
-
Ammonium
NH4
+
Biological N-Fixation (BNF)
- Rhizobium / legume nodules
- Spirochetes/Bacteroidales
- Gram +: Clostridium, Bacillus
- Frankia / Actinorhizal
plant nodules
- Cyanobacteria:
Anabaena, Nostoc,
Trichodesmium
- etc.
Lightning
NO2
-
Nitrosifying bacteria:
Nitrosomonas
Ammonia-oxidizing Archaea
Nitrifying bacteria:
Nitrobacter
Nitrification
Soil bacteria
Urea
CO(NH3)2
Excretion
Herbivory
Denitrification
Pseudomonas,
etc.
Leaching*
*Stemflow, throughfall, litter
Nutrients for mosses, etc.
Leaching
Volcanism

3. Symbioses between plants and N-fixing bacteria
Benefits for plant = fixed N
Benefit for bacteria, carbon/food source, and sometimes protection from
O2
A major force allowing plants to spread across land, invade new habitats
N – availablity is a major factor limiting plant growth in many habitats.

4. Symbioses between plants and N-fixing bacteria
Endophytic symbioses (bacteria live inside plant tissues)
Nodule-forming symbioses
Rhizobium / Legumes
Rhizobium / Parasponia (Cannabaceae)
Frankia / Actinorhizal plants (8 families)
• Fagales (Betulaceae, Casuarinaceae, Myricaceae)
• Rosales (Rosaceae, Rhamnaceae, Eleaganceae)
• Cururbitales (Datiscaceae, Coriariaceae)
Symbioses involving Cyanobacteria
Nonvascular plants: Liverworts, hornworts, mosses
Ferns: Azolla with Anabaena symbiont
Cycads: Coralloid roots
Angiosperms: Gunnera with Nostoc symbiont
Associative symbioses (e.g., Poaceae)
Not present inside plant tissues; rather in rhizosphere
Can significantly increase plant growth/biomass
Legume nodules
Cycad coralloid root

5. Rhizobium – Legume symbioses
Rhizobium, an alpha-proteobacterium (Gram negative)
Legumes: Family Fabaceae (beans, peas, Acacia, Lupinus, Acmispon, Bauhinia, etc.), one
of the largest, most successful angiosperm families: 18,000 spp.
Infection and nodulation:
1. Free-living Rhizobium are attracted to root exudates; attach to root and multiply
2. Symbiotic genes activated in both plant and Rhizobium
3. Bacteria enter root, root cortex cells divide to form nodule.
4. Vascular system forms to supply photosynthates to bacteria, take up ammonium
5. Leghemoglobin delivers O2 to the otherwise anaerobic nodule—responsible for pink
color of nodules
Bauhinia (orchid tree) Acmispon glaber Medicago nodules (pink due to leg-
Hemoglobin)

6. Actinorhizal symbioses
220 spp incl. 8 families form endosymbioses with the actinomycete
bacterium Frankia
Rosales: Rosaceae, Eleanaceae, Rhamnaceae
Cucurbitales: Datiscaceae, Coriariaceae
Fagales: Betulaceae, Casaurinaceae, Myricaceae
Actinomycetes (e.g., Frankia)
Gram positive
Mycelial growth—bind soils in a netlike structure
Very abundant in soils
Very important in C and N cycling
Fix N as free-living bacteria and in plant nodules (unlike Rhizobium)
Metabolically more active in nodules
Vesicles are sites of N-fixation, protect from O2 poisoning
Produce Geosmins—distinctive smell of soils
Warmth in compost piles
Form nodules similar to those in Rhizobium-legume symbiosis
Complex interactions involving many signaling compounds, modification of
plant tissues (= modified lateral roots)
Frankia

7. Actinorhizal symbioses 2
All actinorhizal plants belong to the Rosid I clade and share a common
ancestor with Legumes
100 Mya ancestor evolved basis for evolution of RNS (root nodule symbiosis)
This has evolved independently several times 50 – 60 mya (Doyle, 2011)
Rosales: Rosaceae, Eleanaceae, Rhamnaceae
Cucurbitales: Datiscaceae, Coriariaceae
Fagales: Betulaceae, Casaurinaceae, Myricaceae
Root nodule formation entails complex interactions involving many
signaling compounds, modification of plant tissues (= modified lateral
roots)
Many actinorhizal plants also have mycorhizal symbioses and can grow in
very N-poor soils
Many are pioneer species and colonizers of disturbed areas (e.g., Alnus)
Many are used in restoration, preventing desertification (e.g., Casuarina)

8. Actinorhizal associations 3
Infection process and nodule development
Intracellular infection (e.g., Fagales)
1. Root hairs deformed by “Frankia signals”
2. Hyphae enmesh with root hairs, penetrate root
3. Penetration causes cell divisions in root forming a prenodule
4. Nodule primordium arises from root pericycle
5. Nodule is a modified lateral root. Vesicles form at tips of hyphae
Intercellular infection (e.g., Rosales)
1. No root hair deformation
2. Frankia grow in middle lamella, spread through the apoplast
Mature Nodules are modified lateral roots from pericycle
Multilobed, each lobe with vascular bundle
Periderm, endodermis, expanded cortex.
In Casuarina, plant cell wall lignification = O2 protection (no vesicles)
Frankia nodule

9. Actinorhizal symbioses and Rhizobium-Legume symbiotic
signalling mechanisms likely evolved from (are
homologous with) mycorhizal signaling systems
Arbuscular-Mycorhizal symbioses evolved 400 Ma (Remy et al 1994)
RNS evolved repeatedly starting 60 – 70 Ma (Doyle 1998)

10. Rhizobium – Parasponia (Cannabaceae)
Only non-legume plant known to form nodules with Rhizobium
As in Rhizobium – Legume symbioses, depends on Nod factors secrete from host
plant
Similar ontogeny to actinorhizal plants
As with Actinorhizal plants, nodules are modified lateral roots.
Parasponia are pioneer species in N-poor soils and disturbed habitats.

11. Plant – Cyanobacterial Symbioses
Cyanobacteria (“blue-green algae”)
“Arguably the most important organisms ever to appear on earth” – Andrew
Knoll
Invented oxygenic photosynthesis (= “Oxygen Revolution”) using H2O as an
electron source rather than sulfide (H2S)
Enslavement by a eukaryote led to establishment of plastids in Archaeplastida
(Glaucophytes, Red Algae, Green Algae, Land Plants)
Secondary and tertiary symbiogenesis in Stramenopiles (Brown algae, diatoms),
Alveolates (Dinoflagellates), Excavates (Euglenids), Rhizaria (Chlorachniophytes),
etc
Very common in marine (Prochlorococcus, Synechococcus), freshwater,
terrestrial habitats, in lichens, microbial mats, and in N-fixing symbioses w/ plants
Prochlorococcus, a 0.6 µm marine alga discovered in 1986, and likely the
most abundant organism on the planet, producing ~50% of all atmospheric
O2

12. Plant – Cyanobacterial Symbioses 2
Cyanobacteria involved in N-fixing symbioses with plants commonly belong
to the order Nostocales: Specialized N-fixing cells (heterocysts), resting stage cells
(akinetes)
Short-lived gliding filaments called hormogonia are important for infection
of host plant. Factors released from host plant under N starvation
Increased frequency of heterocysts when in symbiosis with a plant
Cyanobacteria associate with nonvascular plants (mosses, liverworts,
hornworts), ferns, cycads, and angiosperms
Anabaena filaments
Heterocyst
Akinete

13. Plant – Cyanobacterial Symbioses 3
“Bryophytes” (non-vascular plants):
Marchantiophyta (liverworts): 2 spp
Anthoceratophyta (hornworts): All species
Bryophyta: few spp.
Endosymbiont filaments are housed in specialized cavities (auricles in
liverworts; slime cavities in hornworts). Cavities continue to form as
gametophyte grows
Ferns: The aquatic fern Azolla holds Anabaena filaments in specialized cavities in
leaves. Symbiosis may date to 130 Ma
Cyanobiont permanently associated with host during all stages of lifecycle
Used as fertilizer in rice paddies
Azolla with Anabaena

14. Plant – Cyanobacterial Symbioses 3
Cycads:
All of the approximately 150 spp of cycads harbor N-fixing cyanobacteria in
coralloid roots
Coralloid roots arise from lateral roots and subsequently are colonized by
cyanobacterial filaments.
Cycad coralloid roots

15. Plant – Cyanobacterial Symbioses 4
Angiosperms – Gunnera in the Gunneraceae
Cyanobiont (Nostoc) enters the Gunnera stem through specialized glands that
secrete polysaccharide mucilage
Cyanobiont is held intracellularly (unlike in non-vascular plants, Azolla, and
cycads), with filaments occupying most of the host cells (filaments surrounded by
host cytoplasm)
Gunnera

16. Associative N-Fixation
Rhizobacteria: Colonize the rhizosphere
Those that have a positive impact on plant growth are called Plant Growth
Promoting Rhizobacteria (PGPR)
Many are attracted by root exudates, and adhere to roots, sometimes forming
biofilms
Less complex than nodule symbioses, but still require molecular signalling
Occur in several plant groups including Poaceae
Many soil-dwelling diazotrophic bacteria (alpha- and beta- proteobacteria)
identified as rhizobacteria: Acetobacter, Azotobacter, Pseudomonas, etc.
PGPR significantly increase plant height, biomass in wheat, rice, corn
In some PGPR, production of phytohormones may also influence plant
growth
May increase P and Fe availability
Likely suppresses growth of harmful bacteria