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:

BiogeochemistryImmobilizationMineralizationDiazotroph

Atmospheric N2

NitrateNO3

-AmmoniumNH4

+

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:NitrosomonasAmmonia-oxidizing Archaea

Nitrifying bacteria:Nitrobacter

Nitrification

Soil bacteria

UreaCO(NH3)2

Excretion

Herbivory

DenitrificationPseudomonas, etc.

Leaching*

*Stemflow, throughfall, litterNutrients for mosses, etc.

Leaching

Volcanism

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.

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 rhizosphereCan significantly increase plant growth/biomass

Legume nodules

Cycad coralloid root

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 multiply2. Symbiotic genes activated in both plant and Rhizobium3. Bacteria enter root, root cortex cells divide to form nodule. 4. Vascular system forms to supply photosynthates to bacteria, take up ammonium5. 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)

Actinorhizal symbioses

220 spp incl. 8 families form endosymbioses with the actinomycete bacterium Frankia

Rosales: Rosaceae, Eleanaceae, RhamnaceaeCucurbitales: Datiscaceae, CoriariaceaeFagales: Betulaceae, Casaurinaceae, Myricaceae

Actinomycetes (e.g., Frankia)

Gram positiveMycelial growth—bind soils in a netlike structureVery abundant in soilsVery important in C and N cyclingFix N as free-living bacteria and in plant nodules (unlike Rhizobium)

Metabolically more active in nodulesVesicles are sites of N-fixation, protect from O2 poisoning

Produce Geosmins—distinctive smell of soilsWarmth 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

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, RhamnaceaeCucurbitales: Datiscaceae, CoriariaceaeFagales: 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)

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 root3. Penetration causes cell divisions in root forming a prenodule4. Nodule primordium arises from root pericycle5. Nodule is a modified lateral root. Vesicles form at tips of hyphae

Intercellular infection (e.g., Rosales)

1. No root hair deformation2. Frankia grow in middle lamella, spread through the apoplast

Mature Nodules are modified lateral roots from pericycle

Multilobed, each lobe with vascular bundlePeriderm, endodermis, expanded cortex.In Casuarina, plant cell wall lignification = O2 protection (no vesicles)

Frankia nodule

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)

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.

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

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

Plant – Cyanobacterial Symbioses 3

―Bryophytes‖ (non-vascular plants):

Marchantiophyta (liverworts): 2 sppAnthoceratophyta (hornworts): All speciesBryophyta: 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

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

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

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