Microbe – Plant Mutualisms

So You Think You Are AloneFall 2009

Mutualisms

• Repetitive Intro– Mutualisms are interactions between two species

in which both species gain a benefit+ + interactions

Natural Selection

• Natural selection should favor organisms that behave in a way that maximizes their fitness (lifetime reproductive success)

• Natural selection should favor traits that maximizes Benefit – Cost

Mutualisms

• Thus, both species involved in mutualistic relationships should receive benefits that are greater than the costs

Origin of Mutualisms• How did these mutualistic

relationships originate?– John Thompson (1984)

proposed that most mutualistisms arose out of relationships that were originally antagonistic• Pollinators were originally

pollen predators• Fruit dispersers were originally

seed predators

• Over time coevolution caused antagonistic relationships to become mutualistic

Let’s Think About Mutualisms

• You might have a mutualistic relationship with Dairy Queen– You benefit by getting

Blizzards from DQ– DQ benefits by getting

money from you• Everyone Benefits!!!

Costs of Mutualisms

• But there are costs associated with your relationship with Dairy Queen– Costs you money to

buy a blizzard– Costs DQ money to

make and sell a Blizzard

Let’s Think About Units

• The benefits and costs must be measured in some type of unit– If I told you that you would gain a benefit of 200 if

you washed my car would you do it?• It might depend on whether the units of benefit were

cents or dollars

• DQ example– Your costs, DQ’s costs, & DQ’s benefit measured in

dollars– Your benefit measured in enjoyment

Different Units

• It is often difficult to start making cost/benefit decisions when costs and benefits are measured in different units

• How do you convert from one unit to another– E.g. how much money is the benefit that you

receive from eating a blizzard really worth?

DQ and You

• In order for you to buy a Blizzard from DQ – the benefit you gain from eating the blizzard must

be greater than the cost of buying the Blizzard• In order for DQ to sell the Blizzard to you – the benefit they gain must be greater than the

cost for them to make and sell it to you

• Both parties benefit

DQ and You

• DQ is a business designed to maximize their profitsMaximize money they make – money they spend

• How should DQ price the Blizzard?– Imagine that it costs DQ $1.00 to make and sell

the Blizzard.– Thus, the higher they price the Blizzard the more

profit they make• $500 – $1 >>>>> $2.99 - $1

DQ and You

• Thus, DQ would appear to make more profit by charging you $500 for a Blizzard than by charging you $2.99

• Why doesn’t DQ charge so much for a Blizzard?

DQ and You

• In order for DQ to maximize their profit then they have to be aware that if they charge too much for a Blizzard that you won’t buy them– Your Benefit < the cost

• Thus, DQ can’t just gouge their customers because you could choose to shop somewhere else for your desert treats (e.g., Sonic Blast) or get by with no ice cream

You and DQ

• You want to maximize your benefit – cost– The enjoyment you get from eating a Blizzard is

independent of the cost so you should want to pay as little as possible

• However, if you are only willing to pay $0.50 then DQ will go out of business and you won’t be able to buy any more Blizzards.

DQ and You & You and DQ

• Thus, you and Dairy Queen are both trying to maximize your own benefit – cost while keeping the relationship going

• However it is possible for the relationship to break down when either party is no longer receiving more benefit than cost

Selfishness in Mutualisms• Both species in mutualisms are

selected the maximize the benefit – cost

• They shouldn’t worry about the benefit – cost of their partner as long as they can make sure that their partner benefits enough to remain in the partnership

• Thus, organisms might be pretty selfish in these relationships!!!!!!

Nitrogen

• 80 % of atmosphere is N2

• Living organisms can not directly incorporate N2 into biological molecules

• Living organisms need nitrogen– Proteins– DNA– RNA– Other molecules

Nitrogen Fixation

• N2 + 3H2 2NH3

• The reduction of nitrogen gas to ammonia is energy intensive

Nitrogen Fixation

• Nitrogen can be fixed in three ways

• Atmospheric fixation– this occurs spontaneously

due to lightning– a small amount only is fixed

this way.• Industrial fixation –

– the Haber process, which is very energy inefficient

– is used to make nitrogen fertilizers.

Nitrogen Fixation

• Biological fixation – nitrogen-fixing

bacteria • 16 molecules of ATP • a complex set of

enzymes to break the nitrogen bonds so that it can combine with hydrogen

Nitrogen Fixing Bacteria

• Free living– Aerobic - Azotobacter– Anaerobic – Clostridium

• Symbiotic association with plants– Legumes

• e.g. peas, beans• Rhizobium

– Non-legumes• e.g. alder tree• Frankia

Rhizobium

• These bacteria can infect the roots of leguminous plants, leading to the formation of lumps or nodules where the nitrogen fixation takes place.

• About 90% of legume species can become nodulated.

LegumesFamily Fabaceae

Alfalfa

Rhizobia in Root Nodules

In root nodules the nitrogen-fixing form exists as irregular cells called bacteroids which are often club and Y-shaped.

Free Living Rhizobium

• In the soil the bacteria are free living and motile, feeding on the remains of dead organisms. – Free living Rhizobium cannot (or rarely) fix

nitrogen

Origin of Rhizobium/Legume Mutualism

• Don’t really know– An ancestral bacteria might have infected plant

roots• Because the plant benefited from the fixed nitrogen,

rather than excluding the bacteria the plant evolved to facilitate the relationship• Because the bacteria benefited from the sugar supplied

by the plant they might have evolved to facilitate the relationship

Genetics of Nodule Formation

• Specificity genes determine which Rhizobium strain infects which legume. – the pea is the host plant to Rhizobium

leguminosarum biovar viciae– clover acts as host to R. leguminosarum biovar

trifolii.

Genetics of Nodule Formation

• Even if a strain is able to infect a legume, the nodules formed may not be able to fix nitrogen.– ineffective.

• Effective strains induce nitrogen-fixing nodules.– Effectiveness is governed by a different set of genes

in the bacteria from the specificity genes. • Nod genes direct the various stages of

nodulation.

Root Nodules

nodules

Nodule Formation

• Root cells release chemicals into the soil.– encourage the growth of the bacterial population in

the area around the roots (the rhizosphere). • Bacterial cell wall and the root surface interactions

are responsible for the rhizobia recognizing their correct host plant and attaching to the root hairs.

• Flavonoids secreted by the root cells activate the nod genes in the bacteria which then induce nodule formation.

Root Hairs

Root tip

Root hairRoot hairs are surrounded by freeLiving rhizobium

Nodule Formation

• Once bound to the root hair, the bacteria excrete nod factors. These stimulate the hair to curl.

• Rhizobia then invade the root through the hair tip where they induce the formation of an infection thread. This thread is constructed by the root cells and not the bacteria and is formed only in response to infection.

Formation of Infection Thread

Root hair starts to curl formation of an infection thread through which rhizobia enter root cells

Nodule Formation

• The infection thread grows through the root hair cells and penetrates other root cells nearby often with branching of the thread.

• The bacteria multiply within the expanding network of tubes, continuing to produce nod factors which stimulate the root cells to proliferate, eventually forming a root nodule.

Formation of Bacteroids

infection thread spreadsinto adjacent cells bacteroids are released from

the infection thread

Nodule Formation

• Within a week of infection small nodules are visible to the naked eye. Each root nodule is packed with thousands of living Rhizobium bacteria, most of which are in the misshapen form known as bacteroids.

Bacteroids

Nodules

Nitrogenase

• Nitrogenase is the enzyme that catalyses the conversion of nitrogen gas to ammonia in nitrogen-fixing organisms.

• In legumes it only occurs within the bacteroids.

• The reaction requires hydrogen as well as energy from ATP.

Nitrogenase is Oxygen Sensitive

• The nitrogenase complex is sensitive to oxygen, becoming inactivated when exposed to it.

• Free Living Bacteria– have a variety of different mechanisms for

protecting the nitrogenase complex, including high rates of metabolism and physical barriers.

– E.g., Azotobacter overcomes this problem by having the highest rate of respiration of any organism, thus maintaining a low level of oxygen in its cells.

Rhizobia in Nodules• Rhizobia controls oxygen levels in

the nodule with leghaemoglobin. – This red, iron-containing protein

has a similar function to that of haemoglobin; binding to oxygen.

– provides sufficient oxygen for the bacteroids but prevents the accumulation of free oxygen that would destroy the activity of nitrogenase.

• Leghaemoglobin is formed through the interaction of the plant and the rhizobia – neither can produce it alone.

Insides of nodules are red because ofleghaemoglobin

Back to DQ and You

• DQ wants you to buy a Blizzard every day.• However, your need for a Blizzard should vary

seasonally– You gain a great benefit from a Blizzard in the

summer when it is warm– You gain less benefit from a Blizzard in the winter

when it is cold

DQ and You

• Who has control?– DQ advertises like crazy• ‘that’s what I like about TEXAS”

– You control your purse

• Thus, you might like to only be able to buy Blizzards when it is warm.

Rhizobia and Legumes

• Who has control in this relationship?– Do the rhizobia determine how much sugar the

plant send to the nodules? or– Does the plant control how much Nitrogen fixation

takes place by controlling how much sugar it sends to the plant?

• Some early data suggested that the plants had control.

From the Plant’s Perspective

• When does the plant benefit most from having a relationship with rhizobia?– When they need nitrogen

• When do they need more nitrogen?– When they live in soil that has a low nitrogen content

• Therefore we predict, that if plants have control of the system that plants should be more likely to have more nodules in low nitrogen soil.

How would you test this prediction?

• Natural Experiments– Examine the roots of the same species of plants

living in soil with different nitrogen contents• Manipulative Experiments– Experimentally manipulate the soil nitrogen

content • Field or lab

Tests of Prediction

• Many laboratory and field studies in crop plants and some undomesticated legumes have shown Lower nodulation rates with increasing soil nitrogen availability

• This observation suggests that the relative benefits of nodulation decline with increasing abundance of reduced nitrogen, which plants can obtain directly from the soil.

From the Plant’s Perspective

• Plants should benefit less from increasing their access to nitrogen when they are limited by another resources (e.g. phosphorous)

• Thus, predict that if the level of nitrogen in the soil is constant that plants should allow more nodules when the level of other limiting soil nutrients is high

From the Plant’s Perspective

• Legumes also restrict nodulation when inadequate supplies of other nutrients, especially phosphorus, limit plant growth

Conclusions

• The fact that plants reduce nodule formation under conditions of high nitrogen or low phosphorus availability illustrates that plants do not permit unlimited infection by compatible rhizobia.

• Plants have some control over whether or not they are “infected” by rhizobia

From the Plant’s Perspective

• The value of nitrogen to plants varies over time– Once a plant is mature and

has the appropriate concentration of nitrogen in its leaves, increasing the nitrogen content in the plant doesn’t help very much

• When do mature legume plants require more nitrogen?– When they are filling their

seeds.

From the Plant’s Perspective

• Legume seeds are rich in nitrogen– If the plant has control of the system then the plant would

like for the rhizobia to provide more nitrogen during the period of seed filling

– Data shows that to be the case.

• It appears that plants can “turn on” nitrogen fixation by rhizobia – Maybe by increasing the amount of sugar that they send to

the nodules

Phloem

• Xylem moves water and nutrients from roots to the leaves

• Phloem moves sugar around the plant– Plant can control when

and where sugar is moved• Phloem loading and

unloading

But As We Learn More Things Get More Complex

• All Rhizobium are not created equally– Mutualistic rhizobia provide their legume hosts

with nitrogen. • Form nodules & fix nitrogen

– Parasitic rhizobia infect legumes, but fix little or no nitrogen. • Form nodules

– Nonsymbiotic strains are unable to infect legumes at all. • Don’t form nodules

Why have Rhizobium strains with one of these three strategies not displaced

the others?

• We need to think about the costs and benefits associated with the different strategies

Things we need to think about

• Competition– All rhizobia spend some time in the soil, where

they compete for resources. – Symbiotic rhizobia, both mutualistic N2-fixers and

parasitic nonfixers, compete for host plants to colonize

Things we need to think about

• Nitrogen fixation is an energetically expensive process– uses resources that rhizobia could otherwise use for

their own growth and reproduction. • Therefore a good strategy might be to be a

nonfixing bacteria that benefits from the resources provided by the plant– Get the benefit without paying either of the costs

• Bacterial strain benefits but the plant does not.• “cheater”

Benefits of Symbiosis

Founding a nodule can dramatically enhance the reproductive success of rhizobia.• A single rhizobium cell that infects a soybean root may

produce up to 1010 descendants inside a large nodule

• Thus, a rhizobium could produce many more descendants in the soil by founding a nodule than by remaining in the soil.

Stuff Scientists Are Starting to Learn

• First, the chances of infecting a legume may be quite low. – A soil in which a compatible host was last grown five years

before can still contain2.5 x 104 rhizobia per g of soil. – roughly 2 x 109 g/ha of soil in the plow layer would then

contain5 x 1013 rhizobia. – A soybean field with 4 x 105 plants, each forming 100 nodules,

would offer only 4 · 107 opportunities to found a nodule. • Thus, the chances that a given symbiotic rhizobium cell

would successfully found a nodule would be about one in a million.

Stuff Scientists Are Starting to Learn

• Aggregations of rhizobia around a root might attract high populations of predatory protozoa increasing predation risk relative to living in “bulk soil”

• Many rhizobia produce various antibiotics active against other rhizobia – exposure to these antibiotics could be greater for

rhizobia attempting to infect a legume root for those that live in the bulk soil.

Partner Choice

Plants might benefit from being able to choose which strain of rhizobia infects them or by being able to preferentially allocate sugars to nodules that contain mutualistic rhizobium.

• It is well known that crop legumes nodulate non-fixing rhizobia, but allocate few resources to those nodules.

Simms et al. 2006

• Examined wild legume, Lupinus arboreus in experiments where they infected them with 3 strains of rhizobia (low, medium, and high nitrogen fixers)

• Greenhouse experiments showed that plants frequently hosted less cooperative strains– Appears plants can’t control who infects them

• However, the nodules occupied by low fixing strains were smaller. – Suggests they may be able to reduce resources sent to these

nodules

Simms et al. 2006

• Survey of wild-grown plants showed that larger nodules house more high fixing strains– plants may prevent

the spread of exploitation by favoring better cooperators.

Mathematical modelling

Mathematical Modelling

A mechanistic molecular test of the plant-sanction hypothesis in legume–rhizobia mutualism Diana E. Marco et al. 2009

Questions

• Can plants tell whether rhizobia are fixing or non-fixing strains?– Do they preferentially form nodules with fixing

strains?• After nodulation can plants determine which

nodules contain fixing and non-fixing strains?– Do they “sanction” non-fixing strains by sending

them less energy?

Studied Soybeans in the Greenhouse

A mechanistic molecular test of the plant-sanction hypothesis in legume–rhizobia mutualism Diana E. Marco et al. 2009

Split Root Experimental Design

Can Plants Determine Whether or Not a Bacteria is a Fixer or Not Before Nodulation?

• If they can then we predict to see more nodules on roots that have been grown in soil that contains fixing bacteria than on the roots of plants grown in soil that contains non-fixing bacteria

Results- from plants infected by both strains

Closed circles- fixing strain and open circles- non fixing strain

Conclusion

• Because there were equal numbers of nodules on roots grown in soil with fixing and non-fixing bacteria we conclude either– plants are unable to determine whether or not

bacteria are fixing or non-fixing strain or– If they can tell, they are unable to stop non-fixing

strains from forming nodules

Can Plants Determine Which Nodules Are Not Fixing Nitrogen and “Sanction” Them?

• If so, we predict that over time nodules formed by non-fixing bacteria should have fewer bacteria living in them

Split Root Study

• Plants received one type of rhizobia– Predict more rhizobia in nodules of plants infected

with fixing strain than in nodules infected with non-fixing strain

• Plants that received both types of rhizobia– On the same plant, nodules that were infected

with fixing strain should have more rhizobia than nodules infected by non-fixing strain

A & B are two different strains of soybeans

T1 plants- ½ fixing, ½ non-fixingT2 plants- both halves fixingT3 plants- both halves non-fixing

Conclusions

• Plants infected with non-fixing bacteria could tell and stop sending resources to the nodules so the bacteria in the nodules died

• Plants infected by both fixing and non-fixing bacteria could not selective stop sending resources to the non-fixing nodules

• Very Interesting Result!!!!!

References• ELLEN L. SIMMS AND D. LEE TAYLOR. 2002. Partner Choice in Nitrogen-Fixation

Mutualisms of Legumes and Rhizobia. INTEG. AND COMP. BIOL., 42:369–380• E. Toby Kiers, Robert A. Rousseau, Stuart A. Wes & R. Ford Denison. 2003. Host sanctions

and the legume–rhizobium mutualism. NATURE 425, 78-81• R. Ford Denison & E. Toby Kiers. 2004. Lifestyle alternatives for rhizobia: mutualism,

parasitism,and forgoing symbiosis. FEMS Microbiology Letters 237 187–193• Ellen L. Simms, D. Lee Taylor, Joshua Povich, Richard P. Shefferson,J. L. Sachs, M. Urbina

and Y. Tausczik. 2006. An empirical test of partner choice mechanisms in a wild legume–rhizobium interaction. Proc. R. Soc. B. 273, 77–81

• Katy D. Heath and Peter Tiffin. 2007. Context dependence in the coevolution of plant and rhizobial mutualists. Proc. R. Soc. B. 274, 1905–1912

• E. Toby Kiers, Robert A. Rousseau and R. Ford Denison. 2006. Measured sanctions: legume hosts detect quantitative variation in rhizobium cooperation and punish accordingly. Evolutionary Ecology Research, 8: 1077–1086

• Diana E. Marco , Rebeca Pérez-Arnedo, Ángeles Hidalgo-Perea, José Olivares, José E. Ruiz-Sainz and Juan Sanjuán. 2009. A mechanistic molecular test of the plant-sanction hypothesis in legume–rhizobia mutualism. Acta Oecologica 35: 664-667