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Nitrogen fixation

Spontaneous induction of root nodulation

München, 11/26/2014

Plant symbioses with nitrogen-fixing bacteria require genetic reprogramming. An LMU team now shows that the plant can initiate this process spontaneously. The finding could help reduce the use of artificial fertilizer in agriculture.

The root system of the legume Lotus japonica. Overexpression of the fluorescence-labeled receptor kinase SYMRK (pink) leads to spontaneous formation of root nodules (Photo: Martina Ried)

Most terrestrial ecosystems provide insufficient levels of mineral nutrients for the optimal growth of land plants. That is why many millions of tons of fertilizer are applied to agricultural land each year to maintain yields. However, plants have evolved their own methods of acquiring the missing elements, which are based on the formation of symbioses with bacteria or fungi. Most land plants can obtain phosphates from the mutualistic association with so-called arbuscular mycorrhizal fungi. A second type of symbiosis renders plants of the legume family (e.g. beans and peas) independent of nitrogen fertilizer: nitrogen-fixing bacteria called rhizobia – which convert atmospheric nitrogen into ammonium which can be assimilated by plants – colonize specialized structures called nodules that differentiate on legume roots. The team of LMU geneticist Professor Martin Parniske has now demonstrated that root nodules can form spontaneously, in the complete absence of rhizobia, if the root cells produce specific signaling proteins in larger amounts.

Legumes detect the presence of potential symbionts by means of receptor proteins – the so-called receptor kinases NFR1 and NFR5 – found on the root-cell surface, which recognize and bind to chemical signals released by surrounding rhizobia. When an appropriate partner is present, binding of the corresponding signal activates a signal cascade, which alters the pattern of gene expression in root cells. As a result of this reprogramming, the plant forms root nodules. These are in turn colonized by the rhizobia, which then provide the plant with fixed nitrogen. A third surface protein, SYMRK, is essential both for the establishment of arbuscular mycorrhiza and for rootnodule symbiosis. “Up to now,”says Parniske, “it has been unclear whether and how these receptors could be used to optimize the symbiosis, or perhaps even to transfer the capacity for symbiosis to plants that are unable to obtain fixed nitrogen.”

Spontaneous activation of signaling
In the new study, Parniske and his colleagues exploited an insight that has emerged from research on the role of receptor tyrosine kinases in cancer: Under certain circumstances, signal relays can be activated in the absence of their cognate signals. It turns out that the signal serves only to bring receptor molecules together, and the resulting interaction activates the relay. But the same effect can be achieved without any signal if the number of receptor molecules present on the cell surface is sufficiently high. “To find out whether this principle also holds for receptor kinases in plants, we generated transgenic roots in the model plant Lotus japonicus, which overexpressed NFR1, NFR5 or SYMRK,” Parniske explains. “We then asked whether formation of root nodules could be spontaneously induced in such roots.”

And indeed, overproduction of any of the three proteins led to the differentiation of root nodules, even though the roots had not been exposed to rhizobia or to the signal molecules they release. Furthermore, overexpression of SYMRK, but not of NFR1 or NFR5, led to the activation of genes that are required specifically for the symbiosis with arbuscular mycorrhizal fungi. “These results show that overexpression of the right receptor kinases makes it possible to trigger the downstream signaling pathway without application of the stimulus that is normally required,” says Parniske.

A wide range of potential applications
Root nodule symbiosis and arbuscular mycorrhiza both make use of a common genetic program, but the signal pathways diverge at an early point in the signal chain. The observation that NFR1 and NFR5 exclusively activate root nodule symbiosis signaling implies that the decision between root nodule symbiosis and arbuscular mycorrhiza is already made at the level of the receptor proteins. “Since many other genetic programs in plants are also under the control of receptor kinases, their susceptibility to spontaneous activation opens up a wide range of potential applications for this approach,” Parniske points out. “In particular, our findings could make a significant contribution to efforts to transfer rootnodule symbiosis to non-leguminous plants, such as cereals and other agronomically important plants, which are not naturally capable of interacting with nitrogen-fixing bacteria. This would lead to a significant reduction in the demand for nitrogen fertilizers, which are produced by the energy-intensive Haber-Bosch process.”
(eLIFE 2014)         göd