Legumes use calcium signaling to coordinate growth of symbiotic nodules

"Nuclear-localized cyclic nucleotide–gated channels mediate symbiotic calcium oscillations"

SCIENCE 27 MAY 2016 • VOL 352 ISSUE 6289

Animals acquire nitrogen by eating other animals or plants, but this is not a viable option for plants that have to rely on photosynthesis for their food (although insect eating plants like the Venus fly trap remind us that evolution can always find a loophole).

Plants can acquire nitrogen in the form of ammonia from the soil. This ammonia can either be present naturally, or added by humans as fertilizer.

The Haber process allowed production of ammonia on an industrial scale, revolutionizing global agriculture. Chemical ammonia remains one of the most widely used crop fertilizers in the world.

But many wild plants grow in nitrogen-poor soil and it has been known for a long time that certain plants, particularly legumes, can replenish the nitrogen content of the soil after it has been exhausted by other crops.

How is this possible? Certain soil bacteria can take gaseous nitrogen from the air and chemically convert it to ammonia, which can be used by plants for growth. This process is called nitrogen fixation.

Some plants, particularly legumes, are able to form symbiotic associations with these soil bacteria. The soil bacteria recognize the roots of the plant, and infect them. The bacteria extend tiny 'infection threads' into the body of the root. The plant and the bacteria together make a small, swollen structure on the root that is called a nodule.

Symbiotic bacteria invade a plant root and trigger nodule formation (from)

The nodules act as sites of exchange between the plant and nitrogen-fixing soil bacteria. The bacteria take nitrogen gas from the air and turn it into ammonia, which helps the plant grow. In exchange the bacteria receive carbon from the plant in the form of sugar.

How does the plant know how to respond correctly to the nitrogen-fixing bacteria? Plant roots will encounter many parasitic bacteria and fungi in the soil - if they responded to all of them by letting them infect their roots, the plant would not be able to survive. Therefore, both the plant and the symbiotic bacteria need signaling systems to (1) recognize each other, and (2) respond to each other by forming root nodules.

Biology is full of such signaling systems: the mating songs of birds are signals to attract potential mates. The fragrant aroma of flowers comes from the chemicals synthesized by the plant to attract insect pollinators. The smell of a flower may give us joy, but for the plant it acts more like a job posting in a newspaper.

A similar signaling system exists to help legumes and other plants attract the good nitrogen-fixing bacteria, and avoid the bad parasites. To attract good bacteria, plants release a number of chemicals (known as strigolactones and flavenoids) into the soil. The bacteria respond to these chemicals by moving towards the root and secreting a number of chemicals called 'nodulation factors'. These nodulation factors are recognized by the plant, and this triggers the beginning of nodule formation.

The nodulation factors trigger a series of transformations in the cells of the root. They have to change their shape and physiology in a precise manner to give rise to a root nodule. This process is similar to the kind of transformations that occur when a caterpillar transforms into a butterfly, or the body changes that occur when a human child goes through puberty. Just as hormones like estrogen and testosterone tell the tissues of our body to mature into their adult forms, the nodulation factors released by nitrogen-fixing bacteria tell the cells of the plant root to form nodules.

How does the plant know how to respond correctly to the nodulation factors? It is not that the nodulation factors simply cause the roots to swell - they lead to very precise and ordered changes in the cells of the root.

How can the plant perform such complex, organized behavior in response to such a simple chemical signal? In order for this to be possible, the cells of the root must have biological signal transduction systems. 

Most processes in biology rely on some kind of signal transduction systems. Biology is all about ordered responses to changing environments. Living things of all shapes and sizes are constantly absorbing information from the outside world, and they are also constantly responding to this information by changing their behavior - think of a mouse hiding in fear at the sight of an owl's shadow, or a flower bud that blooms only when the season is right.

In all these cases an input signal is received by the organism and converted into some kind of behavioral output. But how?

When you drop a letter into a mailbox your only goal is for it to reach the address you have written down. But this is only possible because of an intricate system of mailmen, post offices, postal codes, systems for sorting mail, etc. that ensure that your letter reaches the correct address. This complex arrangement of people is a kind of signal transduction system: the postal system reads the address you wrote on the envelope and converts this information into a sequence of actions that ends with the delivery of your letter.

Similarly when the nitrogen-fixing bacteria secrete nodulation factors, their only goal is for the plant to start forming root nodules - but for this to be possible the plant must have an appropriate signal transduction system. But instead of mailmen and post offices, the plant cell uses a molecular signaling system composed of things like proteins, sugars, hormones. And calcium ions.

Most of us think of calcium as a structural component of our bones, as a mineral that imparts structural strength rather than as something that has a complex biological function like DNA or estrogen.

But in reality the use of calcium ions as a signaling molecule is widespread throughout biology. Cells often keep reservoirs of calcium ions in special compartments. The correct signal leads to the rapid release of these charged ions, causing a wave of calcium ions to flood the cytoplasm. This free calcium can then interact with other proteins and signal transduction systems to instruct the cell to carry out certain behaviors. 

Alfalfa roots injected with calcium indicator dye;
The dye glows after nodulation factors are added (from here)

Scientists can study calcium signaling in cells by using calcium-sensitive indicator dyes that change colour in the presence of free calcium. By injecting tissues of interest with the appropriate dye, scientists can test if calcium signaling is involved in the response to a particular signal.

In a paper from 1996, the authors injected calcium-sensitive dyes into the root hairs of an alfalfa plant. When they applied nodulation factors to the root hair, the dye changed color to show that the root cells respond to the nodulation factor with a sudden spiking oscillation in their calcium levels.

Calcium imaging of alfalfa root
after applying nodulation factors (from here)

This seemed to suggest that the cells were releasing stores of calcium in response to the nodulation factor, but the precise mechanisms by which this calcium release occurs are not fully understood. 

A recent paper from Giles Oldroyd's lab has filled in some of the missing pieces. By studying specific genetic mutants in a small clover species called Medicago trunculata, the authors were able to identify specific genes that lead to abnormal patterns of calcium oscillations. These genes turn out to be members of a group of genes called ion channels, tiny molecular pores that control the flow of ions between compartments.

Mutations in specific ion channels
cause abnormal calcium signaling (from)

Mutations in three specific calcium channels in Medicago trunculata cause it to have abnormal calcium signaling in response to nodulation factors. Without these three functioning genes, the plant cannot form nodules in response to nitrogen-fixing bacteria.

This paper shows that gated calcium ion channels play a key role in the formation of root nodules by legumes and nitrogen-fixing bacteria. Understanding the biological mechanisms by which nitrogen fixing nodules are formed could help the design of technologies that reduce the use of chemical fertilizers.

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