Genetic modification of flavone biosynthesis in rice enhances biofilm formation of soil diazotrophic bacteria and biological nitrogen fixation

Basics about the biological significance and uptake of nitrogen

Nitrogen is essential for organisms and is found in proteins, nucleic acids and other compounds, such as chlorophyll. Animals take up nitrogen with their food, plants from soil. Plants are not able to use the molecular nitrogen out of the atmosphere (N₂) directly, they rely on other sources such as mineral nitrogen fertiliser, decomposed biomass or certain bacteria that convert atmospheric nitrogen into ammonium nitrogen (NH₄⁺) and thus make it available for plants. Many of the nitrogen-fixing bacteria live in the environment of plant roots and partially in symbioses with them: plants receive available nitrogen from the bacteria and the bacteria receive sugar-containing compounds from the plants.

Conventional agriculture currently uses various nitrogen fertilisers to increase yields. These are, however, expensive, pollute soil and water and contribute to the greenhouse effect. Therefore, approaches are being pursued to make, e.g. nitrogen fertilisation more effective, to bind the excess nitrogen in the soil or to increase the biological nitrogen fixation of bacteria in the soil.

Results of the Yan et al. study

The scientists pursued an indirect approach to increase nitrogen availability in rice plants, i.e. the plants were modified to attract more nitrogen-fixing bacteria. Therefore, they screened for plant compounds that promote the colonisation of nitrogen-fixing bacteria around the plant roots.

The screening process identified substances in rice that stimulate the biofilm formation of an exemplary nitrogen-fixing bacterium (Gluconacetobacter diazotrophicus). Biofilms are stable and protected communities of microorganisms on surfaces which are held together by the slime layers of sugars, proteins and water that they produce. In general, the stimulation of biofilm production was intended to promote the colonisation of nitrogen-fixing bacteria on plant roots and subsequent biological nitrogen fixation.

The scientists tested certain ingredients found in the rice individually to see whether they triggered biofilm formation in cultures of different bacteria. Amongst others, the flavone apigenin promoted biofilm formation in G. diazotrophicus and several other known nitrogen-fixing bacteria.

In order to subsequently increase the amount of apigenin in the rice plant, CRISPR/Cas9 was used to switch off enzymes that degrade the flavone or convert it into other substances. The modified rice plants were cultivated in plant pots for further studies. Their roots were found to contain more apigenin.

Root extracts from the modified rice plants stimulated the biofilm formation of bacterial cultures (G. diazotrophicus) more than those of unmodified rice. Further examination of the whole plant showed that the modified rice plants took up more nitrogen than the unmodified ones. Under limited soil nitrogen conditions, the modified plants had more panicles and their yields increased by 20-30 %. Reduced growth was observed under all conditions as a side effect of the modification.

Examination of soil samples showed that the composition of soil bacteria near the roots of the modified plants changed compared to the unmodified plant. The plant roots recruited more nitrogen-fixing bacteria.

Relevance of the results

The Yan et al. study tested an approach where a change in plant metabolism is intended to attract beneficial bacteria. By switching off the downstream metabolic pathways of the flavone apigenin, it was possible to accumulate the substance in the plant, and thus increase the number of nitrogen-fixing bacteria in the root environment of the plant.

However, interfering with the plant metabolism of apigenin also means that other substances derived from apigenin are produced less or are no longer in the plant. These are mainly further flavones, e.g. luteolin, which is known to play an important role in plant-bacteria interactions – and promote symbioses (Peters et al.). Tricin metabolites, which are also derived from apigenin, are a further example. They play an important role in plant defence against, for example, fungal pathogens and insects, and are incorporated into components of the cell walls (Lam et al.).

Against this backdrop, switching off an existing plant metabolic pathway with the help of CRISPR/Cas9 and then looking solely at the desired effects, does not seem to be a well thought out approach. On the one hand, the modified rice plants could be more susceptible to infections in the field. On the other hand, the composition of the microbiota in the root environment is changed. This may have a positive effect on nitrogen uptake, but it is also conceivable it will have a negative effect on other interactions with microorganisms important for plants, as the plant no longer produces other flavones. There are, for example, also endophytic nitrogen-fixing bacteria in rice plants, i.e. bacteria that live in the internal tissues of plants. These play an important role in the pathogen defence of their host plant (Pittol et al.). The altered composition of the flavones in the rice plant could affect these and other microorganisms and should, therefore, be thoroughly investigated during the development of the plant. Moreover, the susceptibility to pathogens as well as the effects of different environmental conditions on the plant also need to be analysed.

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