LET'S LEARN PLANTS: UG: Plant Physiology

Nitrogen fixation is the process of converting atmospheric nitrogen ( $N_{2}$ ) into ammonia ( $N H_{3}$ ), a form that can be used by living organisms. When this process is carried out by microorganisms, it's known as biological nitrogen fixation. It is the primary way that nitrogen enters the biogeochemical nitrogen cycle. This process is exclusive to prokaryotic organisms, which are often called nitrogen fixers. Nitrogen fixers can be either free-living or live in a symbiotic relationship with a host organism.

Examples of nitrogen fixers:

Symbiotic Nitrogen Fixation
Host Plant	N-fixing symbionts
Leguminous: legumes, Parasponia	Azorhizobium, Bradyrhizobium, Mesorhizobium, Rhizobium, Sinorhizobium
Actinorrhizal: alder (tree), Ceanothus (shrub), Casuarina (tree), Datisca (shrub)	Frankia
Gunnera	Nostoc
Azolla (water fern)	Anabaena
Sugarcane	Acetobacter
Miscanthus	Azospirillum
Free-Living Nitrogen Fixation
Type	N-fixing genera
Cyanobacteria (blue-green algae)	Anabaena, Calothrix, Nostoc
Other bacteria
- Aerobic	Azospirillum, Azotobacter, Beijerinckia, Derxia
- Facultative	Bacillus, Klebsiella
- Anaerobic	Clostridium, Methanococcus (archaebacterium)
- Nonphotosynthetic	Clostridium, Methanococcus (archaebacterium)
- Photosynthetic	Rhodospirillum

Association between host plant and rhizobia

Plant host	Rhizobial symbiont
Parasponia (a nonlegume)	Bradyrhizobium spp.
Soybean (Glycine max)	Bradyrhizobium japonicum (slow-growing type); Sinorhizobium fredii (fast-growing type)
Alfalfa (Medicago sativa)	Sinorhizobium meliloti
Sesbania (aquatic)	Azorhizobium (forms both root and stem nodules)
Bean (Phaseolus)	Rhizobium leguminosarum bv. phaseoli, R. tropici, R. etli
Clover (Trifolium)	Rhizobium leguminosarum bv. trifolii
Pea (Pisum sativum)	Rhizobium leguminosarum bv. vicicae
Aeschynomene (aquatic)	Photosynthetic Bradyrhizobium clade

The Nitrogenase Enzyme Complex

The enzyme complex nitrogenase is responsible for biological nitrogen fixation. This enzyme facilitates the high-energy exchange of electrons required for the reduction of $N_{2}$ to $N H_{3}$ .

Components of Nitrogenase

The nitrogenase enzyme complex consists of two main components:

Fe protein: This is the smaller of the two components and is made of two identical subunits. Each subunit contains an iron-sulfur cluster ( $4 F e$ and $4 S^{2 -}$ ) that participates in redox reactions. The Fe protein is highly sensitive to oxygen and is irreversibly inactivated within 30-45 seconds in its presence.
MoFe protein: This component has four subunits with a total molecular mass of 180 to 235 kDa. Each subunit has two molybdenum-iron-sulfur (Mo-Fe-S) clusters. The MoFe protein is also inactivated by oxygen, but its half-life in air is about 10 minutes.

Mechanism

In the overall reaction, ferredoxin acts as an electron donor to the Fe protein. The Fe protein, in turn, hydrolyzes ATP and reduces the MoFe protein. The MoFe protein can then react with $N_{2}$ and $H^{+}$ to produce ammonia and hydrogen gas. It is an exergonic reaction

Microanaerobic condition is necessary for nitrogen fixation

Because oxygen is a strong electron acceptor that can damage and inactivate nitrogenase, nitrogen fixation must occur under anaerobic (oxygen-free) or microanaerobic (low-oxygen) conditions.

Natural anaerobic environments: Some nitrogen-fixing bacteria, like certain cyanobacteria, function in naturally anaerobic conditions such as flooded fields.
Specialized cells: Cyanobacteria like Anabaena create anaerobic conditions in specialized cells called heterocysts. These cells lack photosystem II, preventing them from generating oxygen.
High respiration rates: Aerobic nitrogen-fixing bacteria like Azotobacter maintain a low oxygen concentration by having high levels of respiration, which consumes oxygen.
Temporal separation: Some organisms like Gloeothece fix nitrogen at night when respiration lowers oxygen levels and carry out oxygen-producing photosynthesis during the day.
Nodule regulation: In symbiotic relationships, plants create a low-oxygen environment within nodules. These nodules contain leghemoglobins which are oxygen-binding proteins that give the nodules a heme-pink color. Leghemoglobins have a high affinity for oxygen and increase its transport to the respiring symbiotic bacteria, effectively reducing the free oxygen concentration to a level (20 to 40 nM) that won't inactivate nitrogenase but is sufficient for respiration.

In Gunnera, nodules are pre-existing stem glands that develop independently of the symbiont. In legumes and actinorhizal plants, the symbionts induce the plant to form root nodules. In grasses, symbionts don't produce root nodules. Instead, they anchor to the root surfaces or live as endophytes inside the apoplasts.

Nodule formation

The symbiotic relationship between legume host plants and rhizobia bacteria is a well-studied example of biological nitrogen fixation. This is not an obligatory relationship; it's established under nitrogen-limited conditions through an elaborate exchange of signals between the plant and the bacterium.

Rhizobia migrates towards the host root hair in response to chemical attractants

1. Colonization of the Rhizosphere

The first step is the migration of rhizobia toward the plant roots. This is a chemotactic response triggered by chemical attractants, such as flavonoids and betaines, secreted by the roots.

Establishing symbiosis requires exchange of signals

2. Nodule Formation

The process of nodule formation involves a complex molecular dialogue:

Signal Exchange: The plant and rhizobia exchange signals. The plant has nodulin genes, and the rhizobia have nodulation (nod) genes.
Nod Genes and their functions:
Common Nod Genes: These genes are found in all rhizobial strains and are responsible for synthesizing the basic structure of the Nod factors. The key common nod genes and their functions are:
- nodA: Catalyzes the addition of a fatty acyl chain to the Nod factor.
- nodB: Acts as a chitin-oligosaccharide deacetylase, removing an acetyl group from the terminal sugar.
- nodC: Is a chitin-oligosaccharide synthase that links N-acetyl-d-glucosamine monomers to form the chitin backbone of the Nod factor.
Host-Specific Nod Genes: These genes differ among rhizobial species and play a vital role in determining which plant species a particular rhizobial strain can infect. They modify the basic Nod factor structure synthesized by the common nod genes.
- nodP, nodQ, nodH, nodF, nodE, and nodL: These genes are involved in modifying the fatty acyl chain or adding specific groups to the sugar moieties of the chitin backbone.
Regulatory NodD Gene: The flavonoids from the plant activate the rhizobial NodD protein. NodD is constitutively expressed and its activation induces the transcription of other nod genes.
Nod Factor Synthesis: The activated nod genes code for proteins that synthesize Nod factors, which are lipochitin oligosaccharide signal molecules. The specific composition of these Nod factors, determined by host-specific nod genes, dictates which plant species can be infected.
Plant Perception: The plant's root hairs have protein kinase receptors with LysM domains that bind to the Nod factors. This binding induces calcium ion oscillations in the root epidermal cells, activating a complex signaling pathway called the symbiotic pathway that leads to changes in gene expression and nodule formation.

3. Infection and Nodule Organogenesis

The physical process of infection and nodule development proceeds as follows:

Root Hair Curling: Nod factors cause the root hair to curl, trapping the rhizobia within the coils.
Infection Thread Formation: The root hair cell wall degrades at the point of infection. An infection thread, an internal tubular extension of the plasma membrane, forms at this site by the fusion of Golgi-derived vesicles. The rhizobia proliferate and are encased within this thread.

Root hair curling and infection thread formation

Nodule Primordium: The cortical cells of the plant root dedifferentiate and begin dividing to form a nodule primordium, which is the precursor to the nodule.
Rhizobia Release: The infection thread elongates through the root hair and cortical cells toward the nodule primordium. When it reaches the primordium, its tip fuses with the host cell's plasma membrane, releasing the bacteria into the cytoplasm. The bacteria are now surrounded by a host-derived membrane, forming an organelle-like structure called a symbiosome.

Rhizobia release

Bacteroid Differentiation: Inside the symbiosome, the bacteria divide and then differentiate into nitrogen-fixing forms called bacteroids.
Vascular System Development: The nodule develops a vascular system to facilitate the exchange of fixed nitrogen (from the bacteroids) for nutrients (from the plant).

Nif and Fix genes

Nitrogen fixation is regulated by specific genes called nitrogen fixation (nif) genes and fixation (fix) genes. These genes encode for proteins that are essential for symbiotic nitrogen fixation in plants and for nitrogen fixation in free-living bacteria.

Nif Genes

The nif genes are the core genes required for nitrogen fixation. They are exclusively found in prokaryotes, such as bacteria and archaea. The products of these genes are directly involved in the nitrogen fixation process, with an important example being the subunits of the nitrogenase enzyme complex. This complex is responsible for catalyzing the reduction of $N_{2}$ to $N H_{3}$ . The nif genes are present in both free-living and symbiotic nitrogen-fixing species.

Fix Genes

The fix genes, or fixation genes, are specifically required for the successful establishment of a functional nitrogen-fixing nodule in a symbiotic relationship. They are found in symbiotic bacteria but have not been identified in free-living nitrogen-fixing bacteria. The products of fix genes include regulatory proteins that monitor and control oxygen levels within the bacteroids (the differentiated form of rhizobia inside a nodule).

Regulation of Nif and Fix Genes

The expression of nif and fix genes is tightly regulated, primarily by oxygen levels. The symbiotic process requires anaerobic or microanaerobic conditions because the nitrogenase enzyme is irreversibly inactivated by oxygen.

FixL and FixJ: A key regulatory mechanism involves the FixL and FixJ proteins. FixL is a hemoprotein kinase that senses oxygen levels. Under low oxygen conditions, FixL acts as a kinase on FixJ. FixJ, in turn, regulates the expression of other transcriptional regulators, namely NifA and FixK.
NifA and FixK: NifA is an activator for the transcription of nif genes and some fix genes.

FixK is another regulator, which may be involved in oxygen sensing.

Outcome: This regulatory cascade ensures that nif gene transcription is activated only when oxygen levels are low, allowing the oxygen-sensitive nitrogenase to function properly. This is crucial for maintaining the symbiotic relationship and successful nitrogen fixation within the nodule.

Nitrogen Metabolism

Click here for slides (PG)

Click here for slides (UG)

Nitrogen Cycle

Nitrogen metabolism is the complex process of how organisms convert and utilize nitrogen. The nitrogen cycle describes the exchange of nitrogen between three main pools: the atmosphere, soil and groundwater, and biomass.

The nitrogen cycle involves several key processes:

Nitrogen Fixation: This is the conversion of atmospheric nitrogen gas (

N_{2}

) into a usable form like ammonia (

N H_{3}

). It can occur through biological, industrial, or electrical means.

Ammonification: In this decomposition process, microorganisms convert organic nitrogen from decaying biomass into ammonia (

N H_{3}

) and ammonium ions (

N H_{4}^{+}

)

. Example: Bacillus vulgaris and Bacillus ramosus

Nitrification: This two-step process converts ammonia to nitrate ( $N O_{3}^{-}$ ). First, bacteria like Nitrosomonas or Nitrococcus oxidize ammonia to nitrite ( $N O_{2}^{-}$ ). Next, Nitrobacter oxidizes the nitrite to nitrate. These bacteria are chemoautotrophs and are known as nitrifying bacteria.

Denitrification: This process converts nitrates and nitrites back into dinitrogen gas ( $N_{2}$ ) and is carried out by microorganisms called denitrifiers, such as Thiobacillus denitrificans.

Nitrate Assimilation: Plants actively absorb nitrate from the soil and convert it into organic nitrogen compounds.

Nitrate Assimilation in Plants

Nitrate assimilation is a two-step process in plants:

Step 1: Reduction of Nitrate to Nitrite

This reaction occurs in the cytosol and is catalyzed by the enzyme nitrate reductase (NR). It involves the transfer of two electrons from an electron donor, typically NADH in most cases or NADPH in nongreen tissues.

Nitrate reductases in higher plants are composed of two identical subunits, each containing three prosthetic groups:

flavin adenine dinucleotide (FAD)
heme
a molybdenum ion complexed to a pterin molecule

FAD accepts two electrons from NAD(P)H, which are then passed to the heme domain and finally to the molybdenum complex to be transferred to nitrate. Molybdenum is crucial for this enzyme's function, and a deficiency in it can lead to nitrate accumulation.

Regulation of Nitrate Reductase

NR synthesis is regulated at transcriptional and translational levels by several factors, including nitrate, light, and carbohydrate accumulation. The enzyme is also subject to posttranslational modification through reversible phosphorylation.

Activation: Light, carbohydrates, and other environmental factors stimulate a protein phosphatase that dephosphorylates a key serine residue on the hinge 1 region of NR enzyme, activating it.
Deactivation: In darkness, a protein kinase stimulated by Mg2+ phosphorylates the same serine residue. This phosphorylation allows the enzyme to interact with a 14-3-3 inhibitor protein, rendering it inactive.

This phosphorylation-based regulation provides a rapid way to control NR activity, happening in minutes compared to the hours it takes for enzyme synthesis or degradation.

Step 2: Reduction of Nitrite to Ammonium

Nitrite is a highly reactive and potentially toxic ion, so it's immediately transported from the cytosol to chloroplasts or plastids where it is reduced to ammonium ( $N H_{4}^{+}$ ) by the enzyme nitrite reductase (NiR). This process requires the transfer of six electrons. The electrons are donated by reduced ferredoxin ( $F_{re d}$ ).

NiR consists of a single polypeptide with two prosthetic groups:

an iron-sulfur cluster ( $F e_{4} S_{4}$ )
a specialized heme

Electrons flow from ferredoxin, through the iron-sulfur cluster and heme, to nitrite. Reduced ferredoxin is derived from the photosynthetic electron transport chain or from NADPH generated in oxidative pentose phosphate pathway in nongreen tissue.

Regulation of Nitrite Reductase

Activation: High nitrate levels or exposure to light induce the transcription of NiR mRNA.
Deactivation: The accumulation of end products, such as asparagine and glutamine, represses this induction.

Ammonia Assimilation

Plant cells quickly convert the ammonium generated from nitrate assimilation into amino acids to prevent toxicity. The main pathway for this conversion is the GS-GOGAT pathway, which involves two key enzymes:

glutamine synthetase (GS)
glutamate synthase (GOGAT)

Step 1: Glutamine Synthetase (GS)

GS combines ammonium with glutamate to form glutamine. This reaction requires ATP and a divalent cation like Mg2+, $M n2+$ $, or C o$ 2+ as a cofactor.

Plants have three classes of GS with different functions and locations:

Cytosolic GS: Found in germinating seeds and vascular bundles, producing glutamine for intercellular nitrogen transport. Its expression isn't regulated by light or carbohydrates.
Plastidal GS: Located in roots, generating amide nitrogen for local consumption. Its expression is regulated by light and carbohydrate levels.
Chloroplastic GS: Found in shoots, where it reassimilates photorespiratory ammonium ( $N H_{4}^{+}$ ). Its expression is also regulated by light and carbohydrate levels.

Step 2: Glutamate Synthase (GOGAT)

GOGAT transfers the amide group from glutamine to 2-oxoglutarate, producing two molecules of glutamate. There are two types of GOGAT in plants:

NADH-GOGAT: Accepts electrons from NADH and is found in the plastids of non-photosynthetic tissues like roots or vascular bundles. It assimilates ammonium absorbed from the soil or glutamine translocated from roots or senescing leaves.

Fd-GOGAT: Accepts electrons from ferredoxin and is found in chloroplasts, where it functions in photorespiratory nitrogen metabolism.

Overall reaction in GS-GOGAT pathway

Alternate Ammonia Assimilation Pathways

Plants can also assimilate ammonia through several alternative pathways.

Glutamate Dehydrogenase (GDH)

This enzyme catalyzes a reversible reaction that can either synthesize or deaminate glutamate. An NADH-dependent form is found in mitochondria, while an NADPH-dependent form is in the chloroplasts of photosynthetic organs.

Transamination

This process incorporates nitrogen from glutamine and glutamate into other amino acids. It involves the transfer of an amino group from one amino acid to a keto acid, producing a new amino acid and a new keto acid. This is a reversible process requiring pyridoxal phosphate (vitamin B6) as a cofactor.

Examples:

Asparagine Synthetase (AS)

AS transfers the amide nitrogen from glutamine to aspartate, producing asparagine and glutamate

. It is found in the cytosol of leaves, roots, and nitrogen-fixing nodules.

Regulation of Ammonium Metabolism

The regulation of ammonium metabolism is vital for maintaining the nitrogen-to-carbon (N:C) ratio in plants.

High light and carbohydrate levels: Under these conditions, AS is inhibited, and plastid GS and Fd-GOGAT are activated. This favors the production of carbon-rich compounds like glutamine and glutamate, which are used for synthesizing new plant materials.
Energy-limited conditions: In these situations, the expression of plastid GS and Fd-GOGAT is reduced, and AS is activated. This leads to more asparagine, an amide that helps in long-distance transport or long-term nitrogen storage.

Amino acid biosynthesis

Humans and most animals cannot synthesize certain essential amino acids and must obtain them from their diet. In contrast, plants can synthesize all 20 standard amino acids. The nitrogen-containing amino group is derived from transamination reactions with glutamine or glutamate. The carbon skeletons for amino acids are derived from intermediates of glycolysis (3-phosphoglycerate, phosphoenolpyruvate, or pyruvate) or the citric acid cycle (2-oxoglutarate or oxaloacetate).