Lecture 34

Question 1

Give 4 examples of symbiotic N2 fixation

Answer 1

1. Cyanobacteria with plants
2. Cyanobacteria with fungi in lichens
3. Actinomycete bacteria with woody plants
4. Rhizobium-legume symbiosis

Question 2

Most legumes are nodulated by […]

Answer 2

Rhizobia

Question 3

Give 3 examples of legumes.

Answer 3

Examples: peas, beans, soybean, clover, alfalfa, peanut, etc.

Question 4

Describe the purpose of the symbiotic relationship between legumes and N2-fixing Rhizobia, including what each component provides to the other.

Answer 4

This relationship allows legumes to grow in N poor soils if they are nodulated by N2-fixing rhizobia. They can supply the plant with all the N they need.

In exchange, the plant supplies the N2-fixing bacteroisd within the nodules with an energy source - C4-dicarboxylic acids.

Question 5

Under what circumstances do rhizobia fix N2?

Answer 5

Only when in symbiotic associated with a specific legume plant host. They will not fix N2 when they are free-living.

Question 6

Describe the specificity of the rhizobia-legume relationship.

Answer 6

Most rhizobia can only nodulate specific, closely-related legumes. There are a few species that can have a very broad host range, but this is rare. There is also only one non-legume genus (Parasponia) that can be noduled by rhizobia.

Question 7

What are the 6 steps involved in root nodule formation?

Answer 7

1. Species-specific plant flavonoid signals
2. Rhizobial response
3. Plant response to specific Nod signal
4. Infection
5. Nodule and bacteroid development
6. Nutrient exchange

Question 8

Describe what happens in the species-specific plant flavonoid signals step of root nodule formation.

Answer 8

The roots of each species of legume exude a unique cocktail of organic compounds - this includes low concentrations of phenolic flavonoid compounds such as flavones and isoflavones.

This specific mix of flavone and isoflavone compounds function as species-specific chemical ID signals.

Question 9

Name the flavones or isoflavones specific to soybean and alfalfa.

Answer 9

Soybean: genistein, daidzein (isoflavones)
Alfalfa: luteolin (flavone)

Question 10

What are the substeps in the rhizobial response step of root nodule formation?

Answer 10

A. Colonization
B. Induction of nod genes and species-specific nod signals

Question 11

Describe how the colonization substep of root nodule formation works.

Answer 11

Rhizobia living in the soil can sense and respond to flavonoid signals of their specific host plant. They won’t respond to signals from non-host plants. They will then colonize the rhizosphere, especially near root hair tips. They will then do quorum sensing to determine if sufficient rhizobia are present for successful nodule formation.

Question 12

How do rhizobia do quorum sensing in the colonization step.

Answer 12

N-acyl homoserine lactone allows quorum sensing to occur. The rhizobia have their own auto-inducer: Acyl-HCL. This will tell the bacteria that there’s enough of them to infect the plant.

Question 13

Describe how the induction of nod genes and species-specific Nod signals substeps occurs.

Answer 13

Rhizobial nod genes will be induced in response to the specific flavonoids of their host plant. The nod genes encode enzymes that synthesize a unique LCO Nod signal or Nod factor.

These unique Nod signals function as species-specific chemical ID response signals that stimulate the specific host plant to initiate root nodule formation.

Question 14

Describe the structure of a Nod signal.

Answer 14

It consists of (1) a short chitin backbone, (2) a fatty acid side chain, and (3) unique chemical ‘decorations’ (such as hydroxyl groups) for host specificity. Overall, it is an oligosaccharide with a fatty acid chain and R groups all over.

Question 15

Give an example of the induction of nod genes in S. meliloti.

Answer 15

The gene regulatory protein NodD1 binds to the alfalfa flavonoid luteolin. NodD1-luteolin then turns on the rest of the nod genes.

Question 16

Describe the specificity involved in Nod factor synthesis (incl. relevant genes).

Answer 16

Each Rhizobium makes a unique signal. Enzymes that synthesize that Nod signal sugar backbone and fatty acid side chain are encoded by common nod genes: nodABC.

Enzymes that decorate Nod signals with species-specific features (sulphation, acylation, etc.) are encoded by host specificity nod genes.

Question 17

Describe the plant response step to the Nod signal.

Answer 17

Plants will only response to the NF made by their specific symbionts. The following 3 events will take place:
1. Root hair curling (shepherd’s crook)
2. De-differentiation of root inner cortex cells
3. Cell division begins nodule formation

Question 18

What are the substeps of infection?

Answer 18

A. Infection thread
B. Contact recognition

Question 19

Describe what happens in the infection thread substep of infection.

Answer 19

Rhizobial cells will penetrate into the crook of a root hair. A tube-like infection thread (IT) will form within the root hair, which contains a polysaccharide matrix. Bacteria grow along the IT until they enter the plant cells.

Question 20

Describe what happens in the contact recognition substep of infection.

Answer 20

The plant recognizes that the bacteria in the IT are the correct species. If it decides that the wrong species are present, it will abort the nodule - this prevents infection by pathogens.

This recognition occurs via perception of specific-specific bacterial cell surface determines: exopolysaccharides (EPS) and lipopolysaccharides (LPS)

Question 21

Describe what happens in the nodule and bacteroid development step of nodule formation.

Answer 21

The rhizobia are enveloped by a ‘peribacteroid’ membrane. The plant and bacterial cells development into the specialized nodule necessary for N2 fixation to occur.

The bacteria within this membrane stop growing, but replicate many copies of their genome. They become terminally-differentiated bacteroids that can synthesize nitrogenase to fix N2.

The plant produces leghemoglobin to facilitate rapid O2 transport to the bacteroids and prevent nitrogenase from O2 damage by binding free O2.

Question 22

Describe what happens in the nutrient exchange step of nodule formation.

Answer 22

The plant does photosynthesis, yielding sugars that are transported to root nodule cells. They get converted into C4-dicarboxylic acids, such as succinate, fumarate, and malate. These acids are the bacteroids’ sole source of energy (NOT used for growth).

This generates energy and reducing power for N2 fixation. In exchange, the bacteroids provide fixed nitrogen to the plant: NH4+ and alanine.

Question 23

Are bacteroids still growing? Explain why or why not.

Answer 23

While free-living rhizobia can use malate as a carbon and energy source, bacteroids can only use it as an energy source. Therefore, malate cannot be used for growth.

This is because bacteroids lack key gluconeogenic and biosynthetic enzymes needed for growth on malate, such as PEP carboxykinase.

The bacteroids are therefore terminally-differentiated and cannot escape the nodule.

Question 24

What benefit do rhizobia derive from the symbiosis?

Answer 24

When nodules die, undifferentiated bacteria will be released. These undifferentiated bacteria persist in the infection threads.

In addition, some strains of rhizobia produce rhizopines, which are nutrients that can feed the rhizobia of the same strain in the rhizosphere.