ComBio special: Nodding acquaintance between legumes and bacteria

Terrestrial plants live in a vast, invisible ocean of nitrogen, the ultimate source of the life-giving macronutrient nitrate. Just over 78 per cent of the atmosphere is nitrogen, but the energy needed to transform the inert gas into bioavailable nitrate limits its supply to the biosphere.

Before the invention of the Haber-Bosch process for synthesising ammonium nitrate in 1912, lightning bolts in thunderstorms and nitrogen-fixing soil microbes were the only natural sources of nitrate for plant growth.

Where most plants sip soluble nitrate from the soil in draught form, one group of plants - the legumes - formed an extraordinary biological compact with nitrogen-fixing rhizobia bacteria that, in return for lodgings and a supply of energy-rich carbohydrates, assured them of a permanent, in-house, on-tap supply of nitrate and phosphate.

Dr Giles Oldroyd and his colleague Dr Allan Downie, of the Department of Disease and Stress Biology at Britain's renowned John Innes Centre for plant and microbiology research in Norwich, have made major contributions to describing the biochemical conversation between plant and microbe that precedes the renewal of their ancient arrangement in every new legume seedling.

Legumes' effectively unlimited access to nitrate is of great interest to crop scientists as the world's population heads towards a peak figure of around nine billion by 2050.

An understanding of the signalling mechanisms that establish the symbiosis between plant and microbe could allow plant geneticists to develop non-legume crops, particularly cereals, with the ability to fix their own nitrogen.

At next week's ComBio 2007 conference in Sydney, Oldroyd, one of the world's leading researchers in legume sybmioses, will describe his findings, and his related research into fungal associations, or mycorrhizae, that extend the reach of terrestrial plant root systems to garner extra nutrients - notably, phosphate - and trace elements from the soil.

Symbiosis

Oldroyd and Downie have shown that the two symbioses involve the induction of a common signalling pathway in plants, strong evidence that nitrogen fixation in legumes is a relatively recent variation on an ancient mycorrhizal theme.

Oldroyd says plant-fungal symbioses may have been the key evolutionary innovation that allowed the first primitive plants to colonise the land 450 million years ago. More than 90 per cent of terrestrial plants form mycorrhizal associations with fungi.

In both mycorrhizal and rhizobial symbioses, it is the plant that initiates the conversation, by secreting phenolic compounds from its roots into the rhizosphere.

Different plant taxa secrete 'customised' arrays of phenolic signals that mediate interactions with their preferred symbionts, explaining at least some of the species-specificity of symbiotic interactions.

Oldroyd's John Innes team works with two model legumes, Lotus japonicus and Medicago truncatula. The secreted flavonoid PHIgalactose appears to be the unique signal that initiates the conversation between the legumes and free-living Rhizobium bacteria.

The legume's biochemical 'wink' elicts a nod in response - it activates genes in the bacterium, resulting in secretion of an extremely potent signalling molecule, the nod (nodulation) factor.

The nod factor in turn triggers cortical cells in the plant's roots to expand and proliferate, forming the distinctive root nodules that will accommodate the nitrogen fixing bacteria.

Oldroyd's team has confirmed the primary role of the nod factor in establishing the symbiosis, by showing that extremely low concentrations of pure nod factor are sufficient to induce nodule formation.

The symbiosis is initiated by an infection-like process in which bacteria attach to the plant's root-hair cells, which curl around, entrapping the bacterial cell.

Leghaemoglobin

Oldroyd says the infection is initiated by a single cell, which then spawns a clonal population of specialised cells called bacteroids that remain in the protected environment of the root nodules for the life of the plant.

When the plant dies, millions of bacteria are released from the decaying nodules into the rhizosphere, ready to repeat the cycle.

Within the nodules, the bacteroids produce a nitrogenase enzyme to reduce atmospheric nitrogen to ammonia, which is exported it to the plant's tissues where it is metabolized to nitrate.

The nodule tissues are pink, due to high concentrations of leghaemoglobin, an iron-rich plant protein that fulfils the same basic function in the plant's tissues as haemoglobin in animal blood.

Leghaemoglobin ferries oxygen to the nodule's microbial tenants at a low, constant tension - any excess would suppress the high-energy, anaerobic activity of nitrogenase.

A diffusion barrier around the nodule also regulates the entry of oxygen and other gases, but permits the import of the photosynthetic product dicarboxylic acid, which fuels the high-energy nitrogen-fixing process.

Fungal symbiosis

Oldroyd's laboratory also runs genetics and cell-biology programs to define the plant and bacterial components of signal transmission and transduction. They have shown that the microbe's nod factor initiates nodule growth by activating with several receptor-like protein kinases in its host's cells.

In June last year, the international journal Nature reported a seminal experiment by Oldroyd's team that offered a glimpse of one of the golden prizes of plant genetics: nitrogen fixation by non-legumes.

By introducing a mutation into one of the genes they identified, a calcium-dependent, receptor-like kinase, they created Medicago plants that developed root nodules spontaneously, in the absence of the bacterial nod signal.

The dominant, gain-of-function mutation suggests the nod factor de-represses the inactive plant gene, inducing complex, downstream gene activity that constructs root nodules - complete with a supply of leghaemoglobin.

Activation of the gene induces pronounced oscillations in calcium levels in the cell nucleus. When calcium spiking is suppressed with calcium chelators, the plants developed no root nodules - evidence that calcium serves as a secondary messenger within the cell nucleus.

Oldroyd says the calcium spikes probably coordinate downstream genes involved in nodule formation, or may organize the hair-root curling that allows Rhizobium to successfully infect the plant.

The leghaemoglobin gene, once thought exclusive to legumes, is now known to be present in higher plants. And legumes were not the only plants to exploit its oxygen-carrying capabilities to support nitrogen-fixing symbionts.

Around two decades ago, researchers discovered that a New Guinea rainforest tree, Parasponia andersonii, a distant cousin of elms (Ulmaceae) independently evolved a nitrogen-fixing symbiosis with rhizobia bacteria - specifically, with Bradyrhizobium.

And members of the Australasian genera, Casuarina and Allocasuarina, also form a nitrogen-fixing symbiosis, complete with root nodules - not with rhizobia, but with a mycorrhizal fungus, Frankia.

Nif complex

The casuarinas and Parasponia are of great interest to researchers like Oldroyd, because they suggest that plants already possess much of the genetic machinery required for nitrogen-fixing symbioses.

Could cereals one day be induced to nodulate and fix their own nitrogen? "I would say it is undoubtedly possible," Oldroyd says.

He says rhizobial symbiosis has arisen three times in legumes, as well as in Parasponia, and the casuarinas' association with Frankia underscores the possibility.

"Each time, you get something similar - nodules with a regulated supply of oxygen, and nitrogen-fixing bacteria and fungi," he says. "Our own research highlights the fact that it's not hugely challenging to make a root nodule, so it should be possible to install the same capability in other non-legumes.

"But it's going to depend on how complex the engineering process is. Our research should indicate how difficult it will be."

Oldroyd says that, armed with a much better understanding of the signalling processes between the plant and the bacterium, his team is planning to test its gene constructs in easily transformable plant species like tobacco and Arabidopsis.

If these species can be induced to produce nodules, the John Innes researchers will attempt the leap to more genetically complex species like cereals.

"Two processes are required," he says. "One is to induce nodule meristem tissue to form nodules containing leghaemoglobin. That's quite easy, because development processes involving the turning-on of auxins and cytokinins at the right time, are conserved across plants.

"The other, more difficult, challenge is to successfully induce bacterial infection, which involves the formation and penetration of infection threads into the nodules."

Oldroyd says the fact that nitrogen-fixing species occur in both alpha- and beta- proteobacteria indicates that the nitrogen-fixing (nif) complex of genes has undergone horizontal gene transfer between microbe species in the distant past.

He says a potential problem with engineered, nitrogen-fixing non-legume crops is that they require large amounts of phosphate to fuel the nitrogen-fixation process, and the world's reserves of relatively cheap rock phosphate are dwindling.

The price of fertilisers will rise as scarcity sets in - but so will the cost of producing ammonium nitrate via the Haber-Bosch process, so nitrogen-fixing crops, and crops with more efficient mycorrhizal associations to increase phosphate, would both benefit global food production.

On the latter issue, Oldroyd says the advent of genetically modified, herbicide-tolerant crops will also benefit crop productivity, because repeated cultivation of the soil to control weeds disrupts the intricate networks of fungal hyphae that supply phospate and micronutrients to crop roots.